serglycin is a theranostic target in nasopharyngeal ... · serglycin is a theranostic target in...

Tumor and Stem Cell Biology

Serglycin Is a Theranostic Target in NasopharyngealCarcinoma that Promotes Metastasis

Xin-Jian Li1, Choon Kiat Ong5, Yun Cao1,2, Yan-Qun Xiang3, Jian-Yong Shao1,2, Aikseng Ooi9, Li-Xia Peng1,Wen-Hua Lu1, Zhongfa Zhang11, David Petillo9, Li Qin4, Ying-Na Bao1, Fang-Jing Zheng1,Claramae Shulyn Chia8, N. Gopalakrishna Iyer6, Tie-Bang Kang1, Yi-Xin Zeng1, Khee Chee Soo7,Jeffrey M. Trent10, Bin Tean Teh5,9, and Chao-Nan Qian1,3,5,10

AbstractNasopharyngeal carcinoma (NPC) is known for its high-metastatic potential. Here we report the identification

of the proteoglycan serglycin as a functionally significant regulator of metastasis in this setting. Comparativegenomic expression profiling of NPC cell line clones with high- and low-metastatic potential revealed theserglycin gene (SRGN) as one of the most upregulated genes in highly metastatic cells. RNAi-mediated inhibitionof serglycin expression blocked serglycin secretion and the invasive motility of highly metastatic cells, reducingmetastatic capacity in vivo. Conversely, serglycin overexpression in poorly metastatic cells increased their motilebehavior and metastatic capacity in vivo. Growth rate was not influenced by serglycin in either highly or poorlymetastatic cells. Secreted but not bacterial recombinant serglycin promoted motile behavior, suggesting acritical role for glycosylation in serglycin activity. Serglycin inhibition was associated with reduced expression ofvimentin but not other epithelial–mesenchymal transition proteins. In clinical specimens, serglycin expressionwas elevated significantly in liver metastases from NPC relative to primary NPC tumors. We evaluated theprognostic value of serglycin by immunohistochemical staining of tissue microarrays from 263 NPC patientsfollowed by multivariate analyses. High serglycin expression in primary NPC was found to be an unfavorableindependent indicator of distant metastasis-free and disease-free survival. Our findings establish that glyco-sylated serglycin regulates NPC metastasis via autocrine and paracrine routes, and that it serves as anindependent prognostic indicator of metastasis-free survival and disease-free survival in NPC patients. CancerRes; 71(8); 3162–72. �2011 AACR.

Introduction

Nasopharyngeal carcinoma (NPC) is a common malignancyin southern China and Southeast Asia (1, 2). NPC has thehighest metastasis rate among head and neck cancers (3–5),with the majority of the patients having metastases to regional

lymph nodes (LN) or even distant organs at the time ofdiagnosis (6). However, the molecular mechanisms underlyingNPC metastasis are poorly understood.

Serglycin is a proteoglycan consisting of a core protein towhich negatively charged glycoaminoglycan (GAG) chains ofeither chondroitin sulfate or heparin are attached (7, 8). Thecore protein containing 158 amino acid residues can be dividedinto 3 domains: a signal peptide domain, an N-terminal domainwith unknown function, and a C-terminal domain (9). Thefunctions of serglycin in various cells depend on the type andsize of the GAG chains decorating the core protein (8, 10–19).

Serglycin mRNA or protein has been detected in normalhematopoietic, endothelial, and embryonic stem cells (11, 14,20–23). Serglycin is thought to be important for homeostasisof positively charged components (e.g., proteases) in storagegranules due to its negatively charged GAG chains (24–28).In cytotoxic lymphocytes or natural killer T cells, a macro-molecular complex of granzyme B and perforin complexedwith serglycin induces the apoptosis of target cells (29–34).Serglycin has been associated with tumorigenesis in acutemyeloid leukemia (AML) and myeloma, and it is found to be aselective marker for distinguishing AML from Philadelphiachromosome-negative chronic myeloproliferative disorders. Itis also highly expressed by multiple myeloma cell lines (35, 36).

Authors' Affiliations: 1State Key Laboratory of Oncology in South China,Departments of 2Pathology and 3Nasopharyngeal Carcinoma, SunYat-sen University Cancer Center, Guangzhou, Guangdong; 4Division ofPharmacoproteomics, Institute of Pharmacy and Pharmacology, Univer-sity of South China, Hengyang, Hunan, China; 5NCCS-VARI TranslationalResearch Program, 6Department of Surgical Oncology, and 7Division ofMedical Sciences, National Cancer Center Singapore; 8Department ofGeneral Surgery, Singapore General Hospital, Singapore; 9Laboratoriesof Cancer Genetics and 10Genome Biology, Van Andel Research Institute(VARI), Grand Rapids, Michigan; and 11Center for Systems and Computa-tional Biology, The Wistar Institute, Philadelphia, Pennsylvania

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

Corresponding Author: Chao-Nan Qian, Department of NasopharyngealCarcinoma, Sun Yat-sen University Cancer Center, 651 Dongfeng EastRoad, Guangzhou, Guangdong 510060, P.R. China. Phone: 86-20-87343457; Fax: 86-20-87343624; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-10-3557

�2011 American Association for Cancer Research.

CancerResearch

Cancer Res; 71(8) April 15, 20113162

Research. on July 26, 2020. © 2011 American Association for Cancercancerres.aacrjournals.org Downloaded from

Published OnlineFirst February 2, 2011; DOI: 10.1158/0008-5472.CAN-10-3557

http://cancerres.aacrjournals.org/

Although the involvement of serglycin in tumormetastasis hasbeen speculated (37), the exact role of serglycin in NPCremains unknown.The epithelial–mesenchymal transition (EMT), a funda-

mental process in embryonic development, is involved inthe metastasis and progression of tumors (38, 39). Activationof the EMT program, accompanied by an increase in themesenchymal marker vimentin and loss of the epithelialmarker E-cadherin, endows carcinoma cells with enhancedmigratory and invasive properties that facilitate disseminationto permissive niches (39).

Materials and Methods

Cell culture and cellular growth rateHuman NPC cell line CNE-2 and its clones (S18, S22, and

S26, cultured in less than 50 passages), and SUNE-1 and itsclone 5-8F were maintained in Dulbecco's modified Eagle'smedium supplemented with 10% FBS at 37�C. Cellular growthcurves were plotted by using the cellular viability valuesassessed by the MTS method (Cell Titer 96 Aqueous OneSolution Cell Proliferation Assay solution; Promega).

In vitro migration and invasion assaysWound healing assays and transwell assays were used to

evaluate the migration and invasion abilities of the cells (40).See the Supplementary Methods for more details.

Detection of serglycin in conditioned mediumA total of 2� 106 cells were plated in 100-mm culture plates

and incubated for 48 hours in a regular medium, then replacedwith a serum-free medium and incubated for an additional 24hours. Ten milliliters of conditioned medium was collectedand concentrated to a volume of 200 mL by using Amicon Ultracentrifuge filters (10 kDa molecular weight cutoff pore size;Millipore). Twenty microliters of the concentrated condi-tioned medium was subjected to SDS-PAGE and blottedwith anti-human serglycin (Cat no. H00005552-M03, Abnova)antibodies.

Human tissues and tissue microarrayAll the human tissue samples were obtained from the

Department of Pathology, Sun Yat-sen University Cancer Cen-ter (SYSUCC), with prior patient consents and the approval ofthe Institutional Clinical Ethics Review Board at SYSUCC. Thetissue microarrays (TMA) contained qualified primary NPCsamples from263 pathologically diagnosed patients at SYSUCCbetween December 30, 1997, and September 6, 2002. Of thesepatients, 199 were men and 64 women, with a median age of 46years (ranging from 17 to 77 years). All patients receivedradiotherapy, with doses of 70 to 74 Gy to the primary tumor,60 to 64 Gy to the involved areas of the neck, and 50 Gy to theuninvolved areas of the neck. For the patients with late-stagedisease (stages III and IV), 2 to 3 cycles of induction orconcurrent platinum-based chemotherapy was given. Thepatients were followed up regularly. TMAs were constructedas described previously (41) and the methodology is describedin the Supplementary Methods.

Histologic evaluation and immunohistochemicalstaining

Mouse lymph nodes were routinely fixed and sectioned at 5mm throughout the lymph nodes. To evaluate the microme-tastases in mouse lymph nodes, one section in every 20sequential sections was selected for hematoxylin and eosinstaining.

For immunohistochemical (IHC) staining of serglycin andvimentin, paraffin-embedded tissues were sectioned at 5 mmand IHC staining was performed as described previously (42).Briefly, the sections were incubated with a rabbit anti-humanserglycin polyclonal antibody (Cat No.: HPA000759, Sigma-Aldrich; working dilution 1:50) or mouse anti-human E-cad-herin monoclonal antibody (Abcam) overnight at 4�C, ormouse anti-human Vimentin monoclonal antibody (Neomar-kers; working dilution 1:100) for 30 minutes at room tem-perature. An EnVision kit (DAKO) was used to detect theprimary antibodies followed by 3,3-diaminobenzidine sub-strate visualization and counterstaining with hematoxylin.The intensity of IHC staining in the tumor cells was scoredindependently by 2 pathologists by using the semiquantitativeIRS (immunoreactive score) scale according to Remmele andStegner (43), which takes into account both the intensity of thecolor reaction (no staining ¼ 0; weak staining ¼ 1; moderatestaining ¼ 2; strong staining ¼ 3) and the percentage ofstained cells (0%¼ 0; 1%–10%¼ 1; 11%–50%¼ 2; 51%–80%¼3; 81%–100% ¼ 4). The average value from the 2 referees wasused as the final score.

Quantitative PCRThe methodology of quantitative PCR is described in the

Supplementary Materials and Methods. The sequences of PCRprimers used for amplification of serglycin and glyceralde-hyde-3-phosphate dehydrogenase (GAPDH) were as follows:GAPDH forward, 50-AAGGTCATCCCTGAGCTGAA-30; GAPDHreverse, 50-TGACAAAGTGGTCGTTGAGG-30; serglycin for-ward, 50-TATCCT ACGCGGAGAGCCAGGTAC-30; serglycinreverse, 50-TTCCGTTAGGAAGCCACTCCC AGATC-30. Theexperiments were performed in triplicate.

Lentiviral transduction studiesCell lines stably expressing either serglycin short hairpin

RNA (shRNA) or a scrambled nontarget shRNA were establi-shed by a BLOCK-iT Lentiviral Pol II miR RNAi system(Invitrogen) according to the manufacturer's instruc-tions. The targets of human serglycin shRNAs are 50-CTGGTTCTGGAATCCTCAGTT-30 and 50-CGCTGCAATC-CAGACAGTAAT-30. See the Supplementary Methods formore details.

ImmunoblottingThe primary antibodies, including mouse anti-human ser-

glycin monoclonal antibody (Abnova), mouse anti-human E-cadherin monoclonal antibody (Abcam), rabbit anti-humanvimentin polyclonal antibody (Cell Signalling Technology),and mouse anti-human b-actin monoclonal antibody (SigmaAldrich) were used at a dilution of 1:1,000. See the Supple-mentary Methods for more details.

Serglycin Regulates Metastasis of Nasopharyngeal Carcinoma

www.aacrjournals.org Cancer Res; 71(8) April 15, 2011 3163




Deglycosylation assayConcentrated conditioned medium containing secreted

serglycin protein was treated with an enzymatic GlycoproteinDeglycosylation Kit (EMD Chemicals) according to the man-ufacturer's instructions. Briefly, 20 mL of concentrated condi-tioned medium, mixed with denaturation buffer (2% SDS, 1mol/L b-mercaptoethanol) and reaction buffer (250 mmol/Lsodium phosphate buffer, pH 7.0), was incubated at 100�C for5 minutes. After adding 1 mL each of N-glycosidase F, a2-3,6,8,9-neuraminidase, endo-a-N-acetylgalactosaminidase,b1,4-galactosidase, and b-N-acetylglucosaminidase, plus 2.5mL of TRITON X-100 (15% solution), the conditioned mediumwas incubated at 37�C for 48 hours. Immunoblots were carriedout to determine the efficiency of serglycin deglycosylation.

Animals and spontaneous lymph node metastasis assayFemale athymic mice between 5 and 6 weeks of age were

obtained from Shanghai Institutes for Biological Sciences(Shanghai, China). All the animal studies were conducted inaccordance with the principles and procedures outlined in theguidelines of Institutional Animal Care and Use Committee at

SYSUCC. Xenograft tumors for genomic expression profilingwere generated by subcutaneous injection of the cancer cellsin the mice. The spontaneous LN metastasis model has beenpublished previously (40). Briefly, a total of 1 � 105 cells wereinjected into the left hind footpad of each mouse to generate aprimary tumor. After 7 to 8 weeks, the popliteal LN of the lefthind foot was collected on the terminal day for routine tissueprocessing.

Genomic expression profilingThe methodology of gene expression profiling is described

in the Supplementary Methods. To identify genes that weredifferentially expressed between high- and low-metastasiscells/xenografts, the log2 transformed expression value ofeach gene in S18 cells/xenograft was subtracted by the meanof log2 transformed expression value of the correspondinggene in the 3 low-metastasis cells/xenografts (i.e., CNE2, S22,S26). The results from this calculation were then sorted; the25 most upregulated and 25 most downregulated genesare presented in Figure 1A and B as heat maps. The geneexpression data for this study have been uploaded to the

CBA

CN

E-2

S22

S26

S18

SERGLYICNSPINK6

SERGLYCINSPINK6

CN

E-2

S22

S26

S18

rgly

cin

evel

CGARNF182CCL26DAB2PSG4MFAP5SPINK5L3AKR1C1VTNSERGLYICN

UGT1A6UGT1A8UGT1A3UGT1A1UGT1A4UGT1A9UGT1A5UGT1A7UGT1A10SERGLYCIN

1

10

100

Rel

ativ

e se

rm

RN

A le

PPP2R2BEPDR1SLC12A3WNT5AKANK4FABP4MYPNSPINK4KRT19CGA

CGAABCG2NNMTDAB2WNT5AFAM102BID2SPINK5L3AKR1C1UGT1A6 1

TXNIPCOL17A1KCNS3FXYD3MAP2K6C21orf63HEG1FYNDIO2

DSC3ABCA12S100A14IRF6SH3BP5AKR1C3C21orf63VTNRNF182 Serglycin

in CM

Serglycin in WCL

ββ-Actin

300 kDa

130 kDa

PPP1R14CTMC4RHBDL1ESRP1TSPAN1CSTADSG3SCELLY6D

LAMC2KCTD12KRT17SCELKCNS3KLK10SERPINB2BNC1SLITRK5

250 kDaIRF6S100A14ST6GALNAC1CLCA2ABP1NDUFA4L2ANKRD22SEPP1SERPINB4

KRT6AKRT14S100A2SYKKRT5SHISA2DSG3LOC642587ST6GALNAC1

Differential expression value: ≤ -5, -2.5, 0, 2.5, ≥5

150 kDa

100 kDa

75 kDa

LOC642587CALB1KRT6A

CALB1S100A8S100A9

100,000

1,000,000

1,000

10,000

D

E

Figure 1. Serglycin expressionand glycosylation in high-metastasis clones. Geneexpression profiling wasperformed on cultured cells of the4 lines (A), and on thecorresponding xenograft tumorsgrown subcutaneously in the nudemice (B). Red and blue,respectively, indicate upregulationand downregulation of a generelative to the average of the 3low-metastasis lines (CNE-2, S22,and S26). C, the mRNA levels ofserglycin (normalized to GAPDH)in the 4 lines were confirmed byquantitative real-time PCR,showing that serglycin expressionwas significantly higher in S18cells than in the other lines.Moreover, high level of serglycinmRNA was also found in anotherhigh-metastasis clone 5-8F incontrast to its parental low-metastasis NPC cell line SUNE-1.Column, mean; error bar, �SD(from triplicates). D, the serglycinprotein levels in these lines asdetermined by Western blotting.E, the concentrated conditionedmedium of S18 cells wasdeglycosylated at 37�C for48 hours by an enzymaticdeglycosylation kit. After partialdeglycosylation, a protein band of80 kDa was detected byimmunoblotting of serglycin, incontrast to the control sample withno enzyme. CM, conditionedmedium; WCL, whole cell lysate.

Li et al.

Cancer Res; 71(8) April 15, 2011 Cancer Research3164




Gene Expression Omnibus, with the accession numberGSE24154.

Statistical methodsSingle comparisons were performed by Student's t test,

Mann–Whitney test, or c2 test (2-tailed; P < 0.05was consideredsignificant). The Spearman correlation test (2-tailed) was usedto calculate the correlation coefficient (r) and P value between

the serglycin and vimentin staining scores, or between theserglycin and E-cadherin staining scores. The median ofthe IHC score value was used as the cutoff value to dividethepatients into high- and low-serglycin expression groups. Thecensoring timedistributionwas estimated by theKaplan–Meiermethod and P values were calculated by log rank analysis.Cox's regression model was used for multivariate survivalanalysis. Predictors were judged to be significant at P < 0.05.

Primary NPC Liver metastases

g s

core

s

BA

erg

lyci

n s

tain

ing

Se

PrimaryNPCs

(n = 48)

Liver metastases from NPC(n = 16)

450

*C

100150200250300350400450

mb

er o

f ce

lls/f

ield

CNE-2

CNE-2 M3**

050

Nu

m

600

800

1,000

1,200

tive

ser

gly

cin

mR

NA

leve

l D

0

200

400

CNE-2 CNE-2 M3

Rel

at m

Figure 2. Serglycin expression is upregulated in liver metastases and in migrated CNE-2 cells. A, IHC staining was used to evaluate the protein expressionlevels of serglycin in primary NPC tissues and in liver metastases from NPC. The ranges of serglycin expression (brown) were shown, with prominentstaining in metastatic lesions. Scale bars, 50 mm. B, statistical analysis (Mann–Whitney test) revealed a significant increase of serglycin expression in livermetastases from NPC relative to expression in primary NPC (*, P < 0.001). C, a 2-chamber assay (transwell assay) with and without Matrigel was usedto evaluate migration and invasion abilities, respectively. Parental CNE-2 cells and CNE-2 M3 cells were studied. Sequential screening successfully selectedthe CNE-2M3 cells that had increased migration and invasion abilities. Column, mean; bars,�SD (from triplicates). *, P < 0.001 relative to parental CNE-2 cells(Student's t test). D, serglycin mRNA levels in CNE-2 and CNE-2 M3 cells were determined by quantitative real-time PCR normalized to GAPDH.Serglycin expression was dramatically upregulated in the CNE-2 M3 cells, consistent with the previous findings.






Results

Serglycin expression is elevated in NPC cells with highermetastasis potential

We have previously isolated and established cellularclones having different metastatic abilities from the par-ental NPC cell line, CNE-2 (40). Among these clones, clone18 (S18) had the highest metastatic ability, whereas clone 22(S22) and clone 26 (S26), and the parental line CNE-2, hadlow-metastatic abilities. To explore the underlying molecu-lar mechanism(s), we performed genomic expression profil-ing of these 4 cell lines from in vitro cultured cells. In thehigh-metastasis clone S18, the serglycin gene (SRGN) wasthe second most highly upregulated (Fig. 1A). Consideringthe potential differences between in vivo and in vitro con-ditions, xenograft tumors from these 4 cell lines were alsocollected for gene expression profiling. Again, serglycin was

the second most upregulated gene in S18 xenograft(Fig. 1B).

To confirm the findings described above, quantitativereal-time PCR was performed to quantify the mRNA levelsof serglycin among the 4 lines, with consistent results(Fig. 1C). Moreover, a high level of serglycin expressionwas also detected in the high-metastasis clone 5-8F isolatedfrom another low-metastasis parental NPC cell line SUNE-1(ref. 44; Fig. 1C). Moreover, S18 and 5-8F cells could secreteserglycin into culture medium, whereas the other low-metastasis cell lines could not (Fig. 1D). The specificity ofthe anti-human serglycin antibody was confirmed andshown in Supplementary Figure 1. After partial deglycosyla-tion of secreted serglycin, a protein with a lower molecularweight (approximately 80 kDa) could be detected (Fig. 1E),confirming the glycosylation of the serglycin proteinsecreted by high-metastasis cells.

1 61.8

in

0 40.6 0.8 1.0 1.2 1.4 1.6

MT

S a

ctiv

ity

(A49

0n

m)

ScrambledCM

WCL20,00030,00040,00050,00060,000

elat

ive

serg

lyci

mR

NA

leve

l

0.0 0.2 0.4

1 2 3 4 5 6

M

Days

SG KD2ββ-Actin

010,000R

800

900 Scrambled

SG KD1

D

A B C

E

mb

led

0.5 mm

300

400

500

600

700 SG KD2

* * # #

Scr

aK

D 1

0

100

200

300

Nu

mb

er o

f S

18 c

ells

/fie

ld

SG

K

D 2

0 h 24 h 36 h

SG

SG KD1

70,000

Figure 3. Suppression of serglycin inhibits the migration and invasion abilities of S18 cells. S18 cells were transduced with lentiviruses expressing scrambledshRNA or shRNAs targeting serglycin at different loci (KD1 and KD2). A, serglycin mRNA levels (normalized to GAPDH) determined by quantitative real-time PCR in the serglycin knockdown cells were significantly reduced. B, serglycin protein levels in both conditioned medium (CM) and whole celllysate (WCL) were determined by immunoblotting. Suppression of serglycin in SG KD1 and SG KD2 cells completely eliminated the secretion of serglycinprotein into the medium. C, suppression of serglycin did not alter the cellular growth rate. D, suppression of serglycin dramatically reduced the migrationability of the cells as determined by a wound-healing assay. Yellow dashed lines denote the margins of the wound. E, a 2-chamber assay was used forfurther evaluation of the invasion/migration ability of the S18 cells. Suppression of serglycin significantly reduced the cells’ migration and invasion abilities.Column, mean; bars, SE (from triplicates). Student's t test was used for the statistical analyses. *, P < 0.001 relative to that of scrambled shRNA; #, P¼ 0.0027for SG KD1 and 0.0061 for SG KD2 when compared with that of scrambled shRNA.

Li et al.





Serglycin is upregulated in liver metastases from NPCand in highly migratory/invasive NPC cellsTo evaluate the clinical relevance of our hypothesis that

serglycin regulates NPC metastasis, we collected archivaltissues of liver metastases from NPC (n ¼ 16) for comparisonwith primary NPC tissues (n ¼ 48). IHC staining showed thatthe protein levels of serglycin were significantly higher in theliver metastases (Fig. 2A and B), implying that serglycin couldbe important in clinical scenarios.To confirm that upregulation of serglycin in highly migra-

tory cells could be repeatedly found, a 2-chamber (Boydenchamber) assay was used to isolate highly migratory cells fromthe parental CNE-2 line. After 3 sequential passages of screen-ing, highly migratory/invasive cells (CNE-1 M3) were isolated(Fig. 2C), and serglycin was found to be highly expressed inthese cells (Fig. 2D).

Lower serglycin secretion suppresses the migration andinvasion of NPC cells without influencing growth rateTo examine the causal role of serglycin in motility of NPC

cells, we engineered cell lines from S18 that stably expressedeither shRNAs targeting serglycin expression (SG KD1 and SGKD2) or a scrambled nontarget shRNA. The suppression ofserglycin expression at the mRNA level was confirmed byquantitative real-time PCR (Fig. 3A). A decrease in secreted

serglycin protein was confirmed via immunoblotting (Fig. 3B).Yet, knocking down serglycin in S18 cells did not alter thegrowth rate of the cells (Fig. 3C), implying that serglycin didnot have a role in controlling cellular growth. The migrationability of S18 cells was significantly suppressed after the loss ofsecreted serglycin (Fig. 3D and E). The invasion ability ofS18 cells was also significantly reduced by knocking downserglycin (Fig. 3E). These results proved that secreted serglycincould promote NPC cell migration and invasion in an auto-crine mode.

Secreted serglycin promotes the motility oflow-metastasis NPC cells

To validate the motility-enhancing activity of serglycin, wegenerated an S26 cell line stably overexpressing serglycin inthe conditioned medium (Fig. 4A), without altering the cel-lular growth (Fig. 4B).

The conditioned medium of S26 cells overexpressingserglycin or the control vector was subjected to 50� con-centration (from 10 mL to 200 mL), and concentrated ser-glycin protein was then confirmed by immunoblotting(Fig. 4C). The concentrated conditioned medium was usedto stimulate migration and invasion by wild-type S26 cells.The number of migrated and invaded cells were significantlyincreased after the stimulation (Fig. 4D and E). These results

Figure 4. Secreted serglycinenhances migration and invasionabilities of low-metastasis S26cells. A, low-metastasis S26 cellswere transduced with lentivirusesexpressing serglycin cDNA or anempty vector as control. Theserglycin expression levels weredetermined by Western blotting.Protein levels of serglycin inconditioned medium (CM) andwhole cell lysate (WCL) wereincreased by stable ectopicexpression of serglycin cDNA inS26 cells. B, S26 cells stablyoverexpressing serglycin cDNA orthe control vector were subjectedto MTS assays. There was nostatistical difference betweenthese 2 cellular populations. C, ahigh level of secreted serglycinwas detected by immunoblottingin the concentrated conditionedmedium of the S26 cells carryingserglycin plasmid. Twenty-fourhours of stimulation of wild-typeS26 cells with the 2 conditionedmedia resulted in an increase ofmigration ability (D) and anincrease of invasion ability (E) ofthe S26 cells. Column, mean; bars� SE (from triplicates). *, P < 0.01with Student's t test.

2.0

y

CBA

0.5

1.0

1.5

MT

S a

ctiv

ity

(A49

0n

m)

Vector

SGCM250 kDa

0.0 1 2 3 4 5 6

M

Days

SGWCL

β-Actin

130 kDa

ED450* *

400

500

600

lls p

er f

ield

300

350

400

s p

er in

sert

100

200

300

Mig

rate

d c

e

150

200

250

nva

ded

cel

ls0

Vector SG

100

Vector SG

In






suggest that serglycin could also promote NPC motility via aparacrine mode, by which secreted serglycin from a rareclone could stimulate the motility of cells unable to secreteserglycin. Interestingly, prokaryotic recombinant serglycinwithout glycosylation modification could not promote

migration of the cells (Supplementary Fig. 2), suggestingthat the glycosylation of serglycin is critical for promotingcellular motility.

Expression of serglycin mediates the metastasis rate ofNPC in vivo

The effect of serglycin in vivo was determined by using anLN metastasis model (40). As expected, serglycin knockdowncells showed a significant reduction in metastasis rate, andserglycin-overexpressing cells showed an increased metastasisrate (Fig. 5). These results confirmed that serglycin wasinvolved in the control of NPC metastasis in vivo.

Serglycin expression is associated with the epithelial–mesenchymal transition

To explore the relationship of serglycin and EMT, IHCstaining of serglycin, vimentin, and E-cadherin was performedin 48 primary human NPC tissue samples. The results indi-cated that serglycin expression level positively correlated tothe expression level of vimentin (Fig. 6A and B), and inverselycorrelated to the expression level of E-cadherin (Fig. 6A andC). To confirm this finding, the protein levels of several EMTmarkers in 4 cellular populations were evaluated. A high levelof secreted serglycin accompanied elevated levels of mesen-chymal markers vimentin, N-cadherin, and fibronectin, and areduced level of epithelial protein E-cadherin in the high-metastasis cells (Fig. 6D). The suppression of serglycinresulted in a lower vimentin level, but did not influence thelevels of other EMT proteins (Fig. 6E). All these data suggestthat serglycin is correlated with EMT by mediating thevimentin level.

High-level serglycin expression is an independent,unfavorable prognostic indicator for NPC

Finally, to evaluate the prognostic values of serglycinexpression, we performed IHC staining for serglycin in a setof tissue microarrays containing 263 NPC samples (Supple-mentary Table 1; Fig. 7A and B). The correlations betweenserglycin expression and clinicopathologic characteristics arepresented in Supplementary Table 2. High serglycin expres-sion was significantly correlated with the occurrence of dis-ease progression or distant metastasis. Multivariate analysesrevealed that a high level of serglycin expression was anindependent, unfavorable prognostic indicator for disease-free survival and distant metastasis-free survival (Supplemen-tary Table 3). The survival curves of disease-free survival anddistant metastasis-free survival are presented in Figure 7Cand D. Taken together, these analyses revealed that highserglycin expression level in NPC significantly correlated withadverse patient outcomes.

Discussion

In this study, we have identified a metastasis-enhancingglycoprotein, serglycin, by using high-throughput gene expres-sion profiling analyses of CNE-2 clones having low- or high-metastasis potential. The first advantage of our approach is tominimize the influences from confounding factors. When

A B

0.5 mm 50 µm

60

P = 0.036

P = 0.032C

5242 43

716 16

10

20

30

40

50

60

Nonmetastasis

Metastasis

Nu

mb

ero

f m

ice

D E

0

10

Scrambled SG KD1 SG KD2

N

50 µm0.5 mm

209

40

50

60

Nonmetastasis

Metastasiser o

f m

ice

P = 0.022F

4252

0

10

20

30

Vector SG overexpression

Nu

mb

e

Serglycin overexpression

Figure 5. In vivo metastasis rates of serglycin knockdown cells andserglycin-overexpressing cells. Subcutaneous injection of S18 cells stablyexpressing serglycin shRNAs (SG KD1 and KD2) or scrambled shRNA intothe left hind footpad of nude mice led to metastasis to the left popliteal LNin some animals. A, image of a popliteal LN 38 days after injection of S18cells expressing a scrambled shRNA. B, enlarged image of the framed areain (A), showing metastatic S18 cells invading the normal lymphoid tissue.C, the proportion of popliteal LN metastasis after inoculation. Knockingdown serglycin in S18 cells significantly reduced the metastasis rate from88.1% (52 of 59) to 72.4% (42 of 58). D, S26 cells stably overexpressingserglycin or a control vector were inoculated into the left hind footpad ofthe mouse. The image shows a metastatic popliteal LN 38 days afterinjection. E, enlarged image of the framed area in (D), showing metastaticserglycin-expressing S26 cells in the subcapsular area of the LN invadingthe normal lymphoid tissue. F, the proportion of popliteal LN metastasisafter inoculation of S26 cells. Overexpression of serglycin in S26 cellssignificantly increased the metastasis rate from 67.7% (42 of 62) to 85.2%(52 of 61).

Li et al.





different cell lines (or tumor tissues) derived from differentpatients are used for comparisons, many confounding factorsmight appear, including gender, race, age, and other diseasebackgrounds. All these factors will be perfectly balanced whenwe compare cellular clones derived from a single patient. Thesecond advantage is to amplify the impact of critical cellularpopulation on metastasis. As reported previously (40), among29 clones isolated from the CNE-2 line, only 2 clones possessedhigh-metastasis ability, with S18 being the most aggressive.Obviously, if we extract the mRNA from the parental line, the

signal from this aggressive clone will be overwhelmed by themajority of low-metastasis populations. However, tumormetastases usually arise from rare clones in the tumor (45).Using the identified culprit clone for comparison can thereforeeasily reveal the key molecules regulating metastasis.

Among the most upregulated genes in S18, serglycin issecond on the list under both in vitro and in vivo conditions,implying the heavy involvement of this glycoprotein in reg-ulating NPC metastasis. To our knowledge, no published datahave directly linked serglycin with tumor metastasis. This

5 7 9

11

enti

n s

core

Case 1 Case 2

gly

cin

n = 47r = 0.305P = 0.037

(1)1 3

-1 1 3 5 7 9 11

Vim

e

Serglycin score

Ser

g

-1

13 5 7 9

11 13

E-c

adh

erin

sco

re

Vim

enti

n n = 46r = - 0.388P = 0.008

-1 1

-1 1 3 5 7 9 11 Serglycin score

-cad

her

in

S18 cells

E-

Vimentin

Serglycin

SerglycinC

M

N-cadherin

Vimentin

Serglycin

E-cadherin

CM

E-cadherin

WC

L Fibronectin

Snail

N-cadherin

N-cadherin

Vimentin

Serglycin

E-cadherin

WC

LC

M

Fibronectin

β-Actin β-Actin

TwistFibronectin

β-Actin

W

A B

C

E

D

Figure 6. Serglycin expression modulates vimentin expression in NPC cells. A, continuous sections of human NPC tissue were subjected to IHC stainingwith antibodies against serglycin, vimentin, and E-cadherin. The high expression of serglycin in the tumor tissue in case 1 was accompanied by an elevatedlevel of vimentin and absence of E-cadherin. Conversely, the low expression of serglycin in the tumor tissue of case 2 was accompanied by absence ofvimentin and an elevated level of E-cadherin. Scale bars, 50 mm. B, using the same methods, 47 primary NPC tissues satisfactorily stained with antibodiesagainst vimentin and serglycin were scored and plotted. A significant positive correlation between serglycin and vimentin was shown. C, For E-cadherinstaining, 46 NPC tissues were qualified for analysis. A significant negative correlation between serglycin and E-cadherin was found. D, immunoblotting ofserglycin and EMT markers showed a high level of secreted serglycin in the conditioned medium of high-metastasis S18 and 5-8F cells, which wasaccompanied by high levels of vimentin, N-cadherin, and fibronectin, and a low level of E-cadherin. Conversely, low expression of secreted serglycin inlow-metastasis cells (CNE-2, S26, and SUNE-1) was accompanied by low levels of vimentin, N-cadherin, and fibronectin, and a high level of E-cadherin.E, knocking down serglycin expression by shRNAs (SG KD1 and SG KD2) dramatically suppressed the vimentin level in a whole cell lysate of S18 cells, butthe levels of other EMT markers were not influenced.






study has discovered that serglycin is involved in regulatingNPC metastasis by promoting the migratory and invasiveabilities of NPC cells via autocrine and paracrine modes, withautocrine mechanism seeming to be more important. Sergly-cin expression can also mediate the level of vimentin, whichnot only is a marker of EMT but also has an important role inregulating cellular migration (46). Moreover, serglycin is anindependent, unfavorable prognostic indicator for distantmetastasis-free survival and disease-free survival in NPCpatients.

As shown in Figure 1A and B, other genes are also highlyexpressed in S18 cells, implying that multiple factors could beinvolved in regulating NPCmetastasis. When serglycin expres-sion was knocked down in high-metastasis S18 cells, a 15%reduction in the metastasis rate was achieved, suggesting thattargeting multiple factors is a hope for complete suppressionof NPC metastasis.

In summary, our study shows that serglycin plays a pivotalrole in regulating NPCmetastasis by way of enhancing cellularmigration, cellular invasiveness, vimentin expression level,and in vivo spread of cancer cells. Moreover, a high level ofserglycin expression can potentially be used to predict shorterdisease-free survival and shorter metastasis-free survival ofNPC patients. Targeting serglycin could be a novel option forprevention of NPC metastasis.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

We thank David Nadziejka, Grand Rapids, Michigan, for critical reading ofthe manuscript.

Grant Support

This work was supported by grants from the State Key Program ofNational Natural Science Foundation of China (Grant 81030043 to C-N.Qian, X-J. Li, Y. Cao, Y-Q. Xiang, L-X. Peng, W-H. Lu, Y-N. Bao, F-J. Zheng),the National High Technology Research and Development Program ofChina (863 Program; Grant 20060102A4002 to X-J. Li, L-X. Peng, W-H.Lu, C-N. Qian, J-Y. Shao, Y-X. Zeng), the Major State Basic ResearchProgram of China (973 Project; Grant 2006CB910104 to Y-X. Zeng), theYouth Science Fund of the National Natural Science Foundation of China(Grant 81000946 to L. Qin), the Van Andel Foundation (A. Ooi, D. Petillo),the National Cancer Center Research Foundation of Singapore (C.K. Ong, A.Ooi, C.S. Chia, N.G. Iyer, B.T. Teh), and the Singapore Millennium Founda-tion (C.K. Ong, B.T. Teh).

The costs of publication of this article were defrayed in part by the paymentof page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received September 30, 2010; revised December 29, 2010; accepted January13, 2011; published OnlineFirst February 2, 2011.

Serglycinlow expression

Pro

po

rtio

n o

f u

nce

nso

red

cas

esD

MF

S p

rob

abili

ty

DF

S p

rob

abili

ty

Follow-up time (months)

BA

C D

1.0

0.8

0.6

0.4

0.2

0.0

0 20

Death event

40 60 80 100120140

0 20 40 60 80 100 120 140 0 20 40 60 80 100 120 140

Serglycin high expression (n = 125)Serglycin high expression (n = 125)

P = 0.001P = 0.003

Serglycin low expression (n = 138)Serglycin low expression (n = 138)1.0

0.8

0.6

0.4

0.2

0.0

1.0

0.8

0.6

0.4

0.2

0.0

Time to metastasis (months)Time to progression (months)

Lo

wm

agn

ific

atio

nH

igh

mag

nif

icat

ion

Serglycinhigh expression

Figure 7. Elevated serglycin levelcorrelates with shorter disease-free survival and distantmetastasis-free survival in NPCpatients. TMA analyses of a cohortof 263 NPC patients diagnosed atM0 were conducted. A, high andlow levels of serglycin proteinexpression in TMA are shownunder both low and highmagnifications of a lightmicroscope. Scale bars, 100 mm.B, the median follow-up time forthis cohort of patients was 83months. C, the disease-freesurvival (DFS) rate wassignificantly higher in the low-serglycin group. D, the distantmetastasis-free survival (DMFS)rate was also significantly higher inthe low-serglycin group.

Li et al.





References1. Wee JT, Ha TC, Loong SL, Qian CN. Is nasopharyngeal cancer really a

"Cantonese cancer"? Chin J Cancer 2010;29:517–26.2. YuMC, Yuan JM. Epidemiology of nasopharyngeal carcinoma. Semin

Cancer Biol 2002;12:421–9.3. Ahmad A, Stefani S. Distant metastases of nasopharyngeal carci-

noma: a study of 256 male patients. J Surg Oncol 1986;33:194–7.

4. Geara FB, Sanguineti G, Tucker SL, Garden AS, Ang KK, MorrisonWH, et al. Carcinoma of the nasopharynx treated by radiotherapyalone: determinants of distant metastasis and survival. RadiotherOncol 1997;43:53–61.

5. Lee AW, Law SC, Foo W, Poon YF, Cheung FK, Chan DK, et al.Retrospective analysis of patients with nasopharyngeal carcinomatreated during 1976–1985: survival after local recurrence. Int J RadiatOncol Biol Phys 1993;26:773–82.

6. Guigay J. Advances in nasopharyngeal carcinoma. Curr Opin Oncol2008;20:264–9.

7. Kolset SO, Zernichow L. Serglycin and secretion in human mono-cytes. Glycoconj J 2008;25:305–11.

8. Pejler G, Abrink M, Wernersson S. Serglycin proteoglycan: regulatingthe storage and activities of hematopoietic proteases. Biofactors2009;35:61–8.

9. Kolset SO, Tveit H. Serglycin–structure and biology. Cell Mol Life Sci2008;65:1073–85.

10. Woulfe DS, Lilliendahl JK, August S, Rauova L, Kowalska MA, AbrinkM, et al. Serglycin proteoglycan deletion induces defects in plateletaggregation and thrombus formation in mice. Blood 2008;111:3458–67.

11. Toyama-Sorimachi N, Kitamura F, Habuchi H, Tobita Y, Kimata K,Miyasaka M. Widespread expression of chondroitin sulfate-type ser-glycins with CD44 binding ability in hematopoietic cells. J Biol Chem1997;272:26714–9.

12. Humphries DE, Wong GW, Friend DS, Gurish MF, Qiu WT, Huang C,et al. Heparin is essential for the storage of specific granule proteasesin mast cells. Nature 1999;400:769–72.

13. Duelli A, Ronnberg E, Waern I, Ringvall M, Kolset SO, Pejler G. Mastcell differentiation and activation is closely linked to expression ofgenes coding for the serglycin proteoglycan core protein and adistinct set of chondroitin sulfate and heparin sulfotransferases.J Immunol 2009;183:7073–83.

14. Schick BP, Gradowski JF, San Antonio JD. Synthesis, secretion, andsubcellular localization of serglycin proteoglycan in human endothelialcells. Blood 2001;97:449–58.

15. Toyama-Sorimachi N,MiyasakaM. A sulfated proteoglycan as a novelligand for CD44. J Dermatol 1994;21:795–801.

16. Toyama-Sorimachi N, Sorimachi H, Tobita Y, Kitamura F, Yagita H,Suzuki K, et al. A novel ligand for CD44 is serglycin, a hematopoieticcell lineage-specific proteoglycan. Possible involvement in lym-phoid cell adherence and activation. J Biol Chem 1995;270:7437–44.

17. Uhlin-Hansen L, Wik T, Kjellen L, Berg E, Forsdahl F, Kolset SO.Proteoglycan metabolism in normal and inflammatory human macro-phages. Blood 1993;82:2880–9.

18. Winberg JO, Kolset SO, Berg E, Uhlin-Hansen L. Macrophagessecrete matrix metalloproteinase 9 covalently linked to the coreprotein of chondroitin sulphate proteoglycans. J Mol Biol 2000;304:669–80.

19. Zernichow L, Dalen KT, Prydz K, Winberg JO, Kolset SO. Secretion ofproteases in serglycin transfected Madin-Darby canine kidney cells.FEBS J 2006;273:536–47.

20. Schick BP, Ho HC, Brodbeck KC, Wrigley CW, Klimas J. Serglycinproteoglycan expression and synthesis in embryonic stem cells.Biochim Biophys Acta 2003;1593:259–67.

21. Elliott JF, Miller CL, Pohajdak B, Talbot D, Helgason CD, BleackleyRC, et al. Induction of a proteoglycan core protein mRNA in mouse Tlymphocytes. Mol Immunol 1993;30:749–54.

22. Kulseth MA, Mustorp SL, Uhlin-Hansen L, Oberg F, Kolset SO.Serglycin expression during monocytic differentiation of U937–1 cells.Glycobiology 1998;8:747–53.

23. Maillet P, Alliel PM, Mitjavila MT, Perin JP, Jolles P, Bonnet F.Expression of the serglycin gene in human leukemic cell lines. Leu-kemia 1992;6:1143–7.

24. Abrink M, Grujic M, Pejler G. Serglycin is essential for maturation ofmast cell secretory granule. J Biol Chem 2004;279:40897–905.

25. Braga T, Grujic M, Lukinius A, Hellman L, Abrink M, Pejler G. Serglycinproteoglycan is required for secretory granule integrity in mucosalmast cells. Biochem J 2007;403:49–57.

26. Henningsson F, Hergeth S, Cortelius R, Abrink M, Pejler G. A role forserglycin proteoglycan in granular retention and processing of mastcell secretory granule components. FEBS J 2006;273:4901–12.

27. Ringvall M, Ronnberg E, Wernersson S, Duelli A, Henningsson F,Abrink M, et al. Serotonin and histamine storage in mast cell secretorygranules is dependent on serglycin proteoglycan. J Allergy ClinImmunol 2008;121:1020–6.

28. Lieberman J. The ABCs of granule-mediated cytotoxicity: new weap-ons in the arsenal. Nat Rev Immunol 2003;3:361–70.

29. Veugelers K, Motyka B, Goping IS, Shostak I, Sawchuk T, BleackleyRC. Granule-mediated killing by granzyme B and perforin requires amannose 6-phosphate receptor and is augmented by cell surfaceheparan sulfate. Mol Biol Cell 2006;17:623–33.

30. Raja SM, Metkar SS, Honing S, Wang B, Russin WA, Pipalia NH, et al.A novel mechanism for protein delivery: granzyme B undergoeselectrostatic exchange from serglycin to target cells. J Biol Chem2005;280:20752–61.

31. Galvin JP, Spaeny-Dekking LH, Wang B, Seth P, Hack CE, FroelichCJ. Apoptosis induced by granzyme B-glycosaminoglycan com-plexes: implications for granule-mediated apoptosis in vivo. J Immu-nol 1999;162:5345–50.

32. Dressel R, Raja SM, Honing S, Seidler T, Froelich CJ, von Figura K,et al. Granzyme-mediated cytotoxicity does not involve the mannose6-phosphate receptors on target cells. J Biol Chem 2004;279:20200–10.

33. Metkar SS, Wang B, Aguilar-Santelises M, Raja SM, Uhlin-Hansen L,Podack E, et al. Cytotoxic cell granule-mediated apoptosis: perforindelivers granzyme B-serglycin complexes into target cells withoutplasma membrane pore formation. Immunity 2002;16:417–28.

34. Raja SM, Wang B, Dantuluri M, Desai UR, Demeler B, Spiegel K, et al.Cytotoxic cell granule-mediated apoptosis. Characterization of themacromolecular complex of granzyme B with serglycin. J Biol Chem2002;277:49523–30.

35. Niemann CU, Kjeldsen L, Ralfkiaer E, Jensen MK, Borregaard N.Serglycin proteoglycan in hematologic malignancies: a marker ofacute myeloid leukemia. Leukemia 2007;21:2406–10.

36. Theocharis AD, Seidel C, Borset M, Dobra K, Baykov V, LabropoulouV, et al. Serglycin constitutively secreted bymyeloma plasma cells is apotent inhibitor of bone mineralization in vitro. J Biol Chem2006;281:35116–28.

37. Schick BP. Serglycin proteoglycan deletion in mouse platelets phy-siological effects and their implications for platelet contributions tothrombosis, inflammation, atherosclerosis, and metastasis. Prog MolBiol Transl Sci; 93:235–87.

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

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

40. Qian CN, Berghuis B, Tsarfaty G, Bruch M, Kort EJ, Ditlev J, et al.Preparing the "soil": the primary tumor induces vasculature reorga-nization in the sentinel lymph node before the arrival of metastaticcancer cells. Cancer Res 2006;66:10365–76.

41. Yao X, Qian CN, Zhang ZF, Tan MH, Kort EJ, Yang XJ, et al. Twodistinct types of blood vessels in clear cell renal cell carcinoma havecontrasting prognostic implications. Clin Cancer Res 2007;13:161–9.

42. Qian CN, Furge KA, Knol J, Huang D, Chen J, Dykema KJ, et al.Activation of the PI3K/AKT pathway induces urothelial carcinoma ofthe renal pelvis: identification in human tumors and confirmation inanimal models. Cancer Res 2009;69:8256–64.

43. Remmele W, Stegner HE. Recommendation for uniform definition ofan immunoreactive score (IRS) for immunohistochemical estrogen






receptor detection (ER-ICA) in breast cancer tissue. Pathologe1987;8:138–40.

44. Song LB, Yan J, Jian SW, Zhang L, Li MZ, Li D, et al. [Molecularmechanisms of tumorgenesis and metastasis in nasopharyngealcarcinoma cell sublines]. Ai Zheng 2002;21:158–62.

45. Chiang AC, Massague J. Molecular basis of metastasis. N Engl J Med2008;359:2814–23.

46. Ivaska J, Pallari HM, Nevo J, Eriksson JE. Novel functions of vimentinin cell adhesion, migration, and signaling. Exp Cell Res 2007;313:2050–62.

Li et al.





2011;71:3162-3172. Published OnlineFirst February 2, 2011.Cancer Res Xin-Jian Li, Choon Kiat Ong, Yun Cao, et al. Carcinoma that Promotes MetastasisSerglycin Is a Theranostic Target in Nasopharyngeal

Updated version

10.1158/0008-5472.CAN-10-3557doi:

Access the most recent version of this article at:

Material

Supplementary

http://cancerres.aacrjournals.org/content/suppl/2011/02/02/0008-5472.CAN-10-3557.DC1

Access the most recent supplemental material at:

Cited articles

http://cancerres.aacrjournals.org/content/71/8/3162.full#ref-list-1

This article cites 45 articles, 17 of which you can access for free at:

Citing articles

http://cancerres.aacrjournals.org/content/71/8/3162.full#related-urls

This article has been cited by 6 HighWire-hosted articles. Access the articles at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

SubscriptionsReprints and

[email protected] at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

Permissions

Rightslink site. (CCC)Click on "Request Permissions" which will take you to the Copyright Clearance Center's

.http://cancerres.aacrjournals.org/content/71/8/3162To request permission to re-use all or part of this article, use this link



http://cancerres.aacrjournals.org/lookup/doi/10.1158/0008-5472.CAN-10-3557

http://cancerres.aacrjournals.org/content/suppl/2011/02/02/0008-5472.CAN-10-3557.DC1

http://cancerres.aacrjournals.org/content/71/8/3162.full#ref-list-1

http://cancerres.aacrjournals.org/content/71/8/3162.full#related-urls

http://cancerres.aacrjournals.org/cgi/alerts

mailto:[email protected]

http://cancerres.aacrjournals.org/content/71/8/3162


serglycin is a theranostic target in nasopharyngeal ... · serglycin is a theranostic target in...

Documents