SPINDLIN1 Promotes Cancer Cell Proliferation through ... · WNT/TCF-4 signaling pathway, and the...

Cancer Genes and Genomics

SPINDLIN1 Promotes Cancer Cell Proliferation throughActivation of WNT/TCF-4 Signaling

Jing-Xue Wang, Quan Zeng, Lin Chen, Ji-Chao Du, Xin-Long Yan, Hong-Feng Yuan, Chao Zhai,Jun-Nian Zhou, Ya-Li Jia, Wen Yue, and Xue-Tao Pei

AbstractSPINDLIN1, a new member of the SPIN/SSTY gene family, was first identified as a gene highly expressed in

ovarian cancer cells. We have previously shown that it is involved in the process of spindle organization andchromosomal stability and plays a role in the development of cancer. Nevertheless, the mechanisms underlying itsoncogenic role are still largely unknown. Here, we first showed that expression of SPINDLIN1 is upregulated inclinical tumors. Ectopic expression of SPINDLIN1 promoted cancer cell proliferation and activated WNT/T-cellfactor (TCF)-4 signaling. The Ser84 and Ser99 amino acids within SPINDLIN1 were further identified as the keyfunctional sites in WNT/TCF-4 signaling activation. Mutation of these two sites of SPINDLIN1 abolished itseffects on promoting WNT/TCF-4 signaling and cancer cell proliferation. We further found that Aurora-A couldinteract with and phosphorylate SPINDLIN1 at its key functional sites, Ser84 and Ser99, suggesting thatphosphorylation of SPINDLIN1 is involved in its oncogenic function. Collectively, these results suggest thatSPINDLIN1, which may be a novel substrate of the Aurora-A kinase, promotes cancer cell growth throughWNT/TCF-4 signaling activation. Mol Cancer Res; 10(3); 326–35. �2012 AACR.

IntroductionSPINDLIN was first reported as an abundant maternal

transcript present in the unfertilized egg and 2-cell, but notthe 8-cell, stage embryo during the transition from oocyte toembryo in the mouse (1). Its name is derived from itsassociation with the meiotic spindle, which occurs as a resultof phosphorylation modification, in a cell-cycle–dependentfashion. In our earlier report on ovarian cancer cells, weidentified human gene SPINDLIN1 as a new member of theSPIN/SSTY gene family. This family is involved in game-togenesis, and the expression of genes in this family can bedetected in early embryos (2).We found that SPINDLIN1 ishighly expressed in human early embryonic tissues, includ-ing ovary and kidney. However, the expression level ofSPINDLIN1 is dramatically decreased and cannot bedetected in any embryonic tissues at 8months. SPINDLIN1is highly expressed inmany kinds ofmalignant tumor tissues,

including ovarian tumors, non–small cell lung cancers, andsome hepatic carcinomas (3).In addition, NIH3T3 cells with ectopic expression of

SPINDLIN1 form colonies in culture and tumors in nudemice, suggesting the oncogenic potential of this gene (4).Wealso reported that SPINDLIN1, a nuclear protein, is relo-cated during cell mitosis and dynamically distributes alongmitotic spindle tubulin, indicating that SPINDLIN1 is anovel spindle protein (5). We also found that ectopicexpression of SPINDLIN1 induces cell-cycle delay in meta-phase and causes chromosome instability (5, 6).The WNT signaling pathway regulates cell fate in a

large number of developmental processes and differenti-ation during embryogenesis (7, 8). In addition, the vitalrole of this pathway in tumorigenesis arouses greatresearch interest (9–11). b-Catenin, stabilized by activa-tion of WNT signaling, is translocated into the nucleus toform nuclear complexes with transcription factors of theT-cell factor/lymphoid enhancer factor (TCF/LEF) familyand subsequently activates several downstream effectorssuch as C-MYC and cyclin D1. These target genes areknown to regulate the cell cycle and contribute to theoncogenic phenotype (12). We believed that uncoveringthe regulators of WNT/TCF-4 signaling would give rise toa better understanding of the potential mechanisms ofcarcinogenesis.Human Aurora-A is commonly amplified in epithelial

malignant tumors, andmore than 50% of ovarian and breastcancers show enhanced activation of this kinase (13, 14). Ithas been shown that rodent fibroblasts transfected withAurora-A form tumors in nude mice, indicating that

Authors' Affiliation: Stem Cell and Regenerative Medicine Laboratory,Beijing Institute of Transfusion Medicine, Beijing, China

Note: Supplementary data for this article are available at Molecular CancerResearch Online (http://mcr.aacrjournals.org/).

J.-X. Wang and Q. Zeng contributed equally to this work.

Corresponding Authors: Wen Yue and Xue-Tao Pei, Stem Cell andRegenerative Medicine Laboratory, Beijing Institute of Transfusion Med-icine, 27 Taiping Road, Beijing 100850, China. Phone: 8610-68164807;Fax: 8610-68167357; E-mail: [email protected] [email protected]

doi: 10.1158/1541-7786.MCR-11-0440

�2012 American Association for Cancer Research.

MolecularCancer

Research

Mol Cancer Res; 10(3) March 2012326

on September 11, 2020. © 2012 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from

Published OnlineFirst January 18, 2012; DOI: 10.1158/1541-7786.MCR-11-0440

http://mcr.aacrjournals.org/

Aurora-A is a cancer susceptibility gene (15). In addition,some studies have shown that Aurora-A plays an essentialrole in chromosome segregation and cell division (16, 17).Nevertheless, the underlyingmechanism bywhich Aurora-Apromotes tumorigenesis remains elusive. Identification ofthe substrates and further exploration of the signalingpathway linking Aurora-A to other key factors regulatingcell growth will provide new insight into understandingtumorigenesis.Here, we further show that SPINDLIN1 is highly

expressed in ovarian cancer tissues and also promotes cancercell proliferation and tumor growth. Most importantly,SPINDLIN1 is found to function as an activator ofWNT/TCF-4 signaling and promotes the expression of theWNT/TCF-4 targets, C-MYC, cyclin D1, and Axin2.Mutation of the 84 and 99 amino acid sites on SPINDLIN1could largely abolish its effects onWNT/TCF-4 activity andcancer cell proliferation, suggesting that SPINDLIN1 maypromote cancer cell growth through the activation ofWNT/TCF-4 signaling. Further analysis suggests that the keyfunctional sites of SPINDLIN1, amino acids Ser84 andSer99, may be candidate phosphorylation sites for Aurora-A.Taken together, our results suggest that SPINDLIN1promotes cancer cell proliferation by activating theWNT/TCF-4 signaling pathway, and the phosphorylationof Ser84 and Ser99 of SPINDLIN1 may be involved in itseffect on WNT/TCF-4 activity.

Materials and MethodsImmunohistochemistryImmunohistochemistry was conducted on microarray

slides containing 53 ovarian cancer tissues and 4 normaltissues obtained from Chaoying Biotechnology. Another 10cancer-free ovarian tissues were obtained from 307 Hospital(Beijing, China). Tissue sections were deparaffinized bysubmerging slides in xylene, rehydrated in decreasing con-centrations of ethanol, and boiled twice in 0.1 mol/L citratebuffer antigen retrieval solution (pH6.0) for 5minutes (eachtime). Staining was conducted using rabbit anti-SPIN-DLIN1 antibodies. Signals were detected using the Vectas-tain Elite ABC Kit (Vector Laboratories). Hematoxylin wasused for counterstaining.

Plasmid clonesTo generate Aurora-A and SPINDLIN1 expression vec-

tors, GFP-Aurora-A was made by cloning its cDNA intopEGFP-C1. C-MYC- and GFP-SPINDLIN1 were con-structed by inserting the open reading frame region ofSPINDLIN1 into pcDNA3.1/MYC-HIS(�) and pEGFP-C1 vector, respectively. For lentivirus generation, theC-MYC–tagged human SPINDLIN1 coding sequence wascloned into the pBPLV vector. To purify the protein frombacterial cells, full-length and 10 GST-SPINDLIN1 con-structs (1, aa 1–59; 2, aa 51–83; 3, aa 51–94; 4, aa 61–94; 5,aa 85–105; 6, aa 85–138; 7, aa 100–138; 8, aa 123–186; 9,aa 175–237; and 10, aa 544–647) were made by cloning thecorresponding cDNAs into the pGEX-4T-1 vector. For

expression of mutant SPINDLIN1 in mammalian cells,wild-type plEGFP-C1-SPINDLIN1 was constructed, andmutants (SPINDLIN1 S84A, S85A, S96A, S99A, and S84/99A) were generated using the QuikChange Site-DirectedMutagenesis Kit (Merck).

Cell culture and transfectionHeLa, A549, H1299, and HEK293T cell lines were

purchased from American Type Culture Collection. HeLaand HEK293T cells were cultured in Dulbecco's ModifiedEagle's Medium (DMEM), and A549 andH1299 cells weregrown in RPMI-1640 medium (Sigma). Both media weresupplemented with 10% FBS (Hyclone), 100 U/mL pen-icillin, and 100 mg/mL streptomycin, and all cells weremaintained at 37�C in 5% CO2. Transfections of the cellswere conducted using Lipofectamine 2000 (Invitrogen)following the manufacturer's instructions.

AntibodiesRabbit polyclonal anti-SPINDLIN1 antibody was

prepared by the Stem Cell and Regenerative MedicineLaboratory, Beijing Institute of TransfusionMedicine (Beij-ing, China). In addition, we used rabbit monoclonal anti-Aurora-A antibody (Cell Signaling Technology), mousemonoclonal anti-C-MYC (9E10) antibody (Santa CruzBiotechnology), mouse monoclonal anti-cyclin D1 (A-12)antibody (Santa Cruz Biotechnology), mouse monoclonalanti-GFP antibody (Proteintech Group), rabbit polyclonalanti-glutathione S-transferase (GST) antibody (ProteintechGroup), and rabbit polyclonal anti-b-actin antibody (SantaCruz Biotechnology).

Colony-forming assayHeLa cells were stably transfected with expression vectors

encoding wild-type SPINDLIN1 or empty vector (pBPLV)using a lentiviral packaging system. The independent cloneswere cultured and 3 SPINDLIN1 clones (SC1, SC2, SC3) aswell as 2 control clones (CC1 andCC2) were used for colonyformation assay. The cells were plated at 500 cells per well in6-well plates and the culture medium was replaced every 3days. At the end of the incubation, the cells were stainedwithcrystal violet and colonies were counted. Each sample wasrepeated in 3 wells.

Cell counting kit-8 cell proliferation assayViable cells were examined using the CCK-8 Assay

(Dojindo). HeLa cells transfected with siRNA-SPINDLIN1or control oligonucleotide were seeded into 96-well plates ata density of 1 � 103 per well and cultured in DMEMcontaining 10% FBS. The medium was exchanged for 100mLDMEMwith 10 mL cell counting kit-8 (CCK-8) reagentand incubated at 37�C for 1 hour. Absorbance wasmeasuredat 450 nm every day over 7 days. Each sample was repeated in6 wells.

Tumorigenicity assayNu/nu mice were purchased from the laboratory animal

center at the Academy of Military Medical Sciences, and

SPINDLIN1 Promotes Cancer Cell Proliferation

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the experiments were carried out in accordance withinstitutional guidelines for laboratory animals. Four clonesof HeLa cells stably overexpressing SPINDLIN1 (SC1,SC2, SC3 and SC4) and 2 clones of control cells (CC1and CC2) were independently implanted into 4 nu/numice. The cells were suspended in PBS, and 3 � 106 cells/200 mL were injected subcutaneously into either flank ofeach mouse. Every 2 or 3 days, tumor diameters weremeasured, and tumor volumes were calculated usingthe formula: a � b2/2 (where a is the largest and b isthe smallest tumor diameter). One month after injection,the mice were sacrificed, the tumors were removed, andtheir weights were recorded.

RNA interferenceAn siRNA sequence used for depleting SPINDLIN1 was

purchased from Sigma-Aldrich. The following targetsequences were chosen for RNA interference: 50-GATT-CAGCATGGGTGGAAA-30. An oligonucleotide sequencewas used as a negative control (Sigma-Aldrich).

In vitro invasion assayHeLa or A549 cells were transfected with pcDNA3.1-

MYC/HIS(�)-SPINDLIN1 or pcDNA3.1-MYC/HIS(�).Matrigel (BD Biosciences), diluted 1:6, was added to eachMillicell Hanging Cell Culture Insert (PET membrane,8.0 mm pore size) and polymerized at 37�C for 4 hours.Cells were added to the upper chamber without FBS, and thelower chamber was filled with the culture medium contain-ing 1.5% FBS. After incubation for 36 hours, the cells on theMatrigel-coated side of the membrane were removed, andcells on the other side were stained with crystal violet. Thecells migrating through the membrane were counted in 5randomly selected fields for each sample, and each samplewas repeated in 3 wells.

Luciferase reporter assayA549 and H1299 cells were cotransfected using Lipofec-

tamine 2000 with the TCF/b-catenin–responsive reporterplasmid, pTOPFLASH, and pcDNA3.1-MYC/HIS(�)-SP-INDLIN1, or the corresponding empty vector pcDNA3.1-MYC/HIS(�), as well as with the Renilla reference plasmidpRL-null. The control reporter pFOPFLASH transfectedwith pcDNA3.1-MYC/HIS(�)-SPINDLIN1 was used toexclude any nonspecific effects. After transfection for 36hours, cells were lysed and then luciferase activity was mea-sured using a Dual-Luciferase Reporter Assay system (Pro-mega). The procedures followed the manufacturer's protocol.The results were analyzed using SOFTMAX PRO SOFT-WARE (MolecularDevices), and each samplewas analyzed intriplicate. Luciferase activity results were normalized toRenillaactivity and expressed as the ratio relative to control plasmidactivity.

ImmunofluorescenceHeLa cells plated on coverslips were transfected with

pEGFP-C1-SPINDLIN1 for 24 hours. The cells were trea-ted with 1% Triton X-100, followed by incubation with

10% goat serum for 30 minutes. After incubation with theAurora-A antibody (at a dilution of 1:25) overnight at 4�C,the cells were washed and incubated with TRITC-conju-gated goat anti-rabbit IgG diluted 1:50, for 30 minutesat 37�C. After washing, the cells were finally stained with40,6-diamidino-2-phenylindole (DAPI; 2 mg/mL; Sigma).Microscopic images were acquired using Zeiss LSM 510META system with a 40� oil objective (Karl Zeiss).

Immunoprecipitation and immunoblot analysisHeLa cells were lysed in radioimmunoprecipitation assay

(RIPA) buffer (0.5% sodium deoxycholate, 0.1% SDS, and1% NP-40) containing protease inhibitor cocktail (Calbio-chem). Approximately, 500 mg of total cellular protein wasincubated with 10mL of primary antibody for 1 hour at 4�C.Then, 20 mL of Protein A/G PLUS-Agarose (Santa CruzBiotechnology) was added, and the samples incubated over-night at 4�C. For immunoblot analysis, we followed thepreviously described methods (2), using anti-Aurora-A andanti-C-MYC antibodies.

GST pull-down assayGST andGST fusion proteins were induced in Escherichia

coli strain BL21 (DE3) by addition of 1 mmol/L isopropyl-b-D-1-thiogalactopyranoside (IPTG), and the cultures weremaintained at 16�C for 4 hours. The proteins were purifiedusing Glutathione Sepharose 4B (Amersham Bioscience)beads according to the manufacturer's instructions. HeLacells transfected with pEGFP-C1-Aurora-A were harvestedafter 24 hours, and the lysates were incubated with purifiedGST fusion protein bound to glutathione-agarose beads at4�C for 4 hours. Then, the mixture was then boiled, andeluted proteins were separated by SDS-PAGE followed byimmunoblot analysis using anti-Aurora-A and anti-GSTantibodies.

Reverse transcription PCRTotal RNA was extracted with TRIzol reagent (Invitro-

gen) following the recommended protocol. The first strandof cDNA was synthesized using oligo(dT) or a randomprimer (TaKaRa) and then reverse transcribed by M-MLV(TaKaRa). PCR was carried out in a final volume of 20 mLusing rTaq DNA-polymerase (TaKaRa). The PCR cycleconditions were 95�C for 5minutes, followed by 35 cycles of95�C for 30 seconds, 55�C for 30 seconds, and 72�C for 30seconds, finishing with 7minutes at 72�C. b-Actin was usedas an internal standard. The PCR products were visualized in1.2% agarose gels by staining with Gel Green (Biotium).The primers for the PCR were as follows:

Forward, 50-GGTGCCTGTAAATCCTTC-30

Reverse, 50-AACTTCTCCTGGTTCCCT-30(SPINDLIN1);

Forward, 50-TACCCTCTCAACGACAGCAG-30

Reverse, 50-TCTTGACATTCTCCTCGGTG-30(C-MYC);

Forward, 50-GCGAGGAACAGAAGTGCG-30

Wang et al.

Mol Cancer Res; 10(3) March 2012 Molecular Cancer Research328




Reverse, 50-AGGCGGTAGTAGGACAGGAA-30 (cyclinD1);

Forward, 50-GTGAGGTCCACGGAAACTGT-30

Reverse, 50-GTGGGTTCTCGGGAAATGA-30 (Axin2)

In vitro kinase assayHeLa cells were transfected with the Aurora-A expression

vector using Lipofectamine 2000. Cells were collected 36hours later and lysed. Aurora-A was immunoprecipitated byProtein A/G PLUS-Agarose. GST and GST-SPINDLIN1mutant proteins were expressed in bacteria and purified. Theimmunoprecipitates were incubated with different GSTfusion proteins and labeled with [g32-P]ATP by the methoddescribed previously (18). The proteins were separated bySDS-PAGE, and the gels were analyzed by autoradiography.

ResultsSPINDLIN1 is highly expressed in ovarian cancer tissuesTo analyze SPINDLIN1 expression in ovarian cancer

tissues, we conducted immunohistochemical analysis usingthe purified SPINDLIN1 antibody. The samples wereobtained from a microarray containing 53 different typesof ovarian cancer tissues, including serous papillary cysta-denocarcinoma, adenocarcinoma, squamous cell carcinoma,and dysgerminoma. Fourteen nontumor ovarian tissues werealso included in the analysis for comparison. The resultsindicated that SPINDLIN1 ismainly expressed in cancer cellnuclei [Fig. 1A (ii–iv)], whereas no expression was found innormal tissues [Fig. 1A(i)]. The statistical data are shownin Fig. 1B. Among grade I cancer tissues, none of the 6samples were positive for SPINDLIN1. In regard to grade IIand grade III/IV tissues, high expression of SPINDLIN1wasobserved in 12 of 23 and 11 of 24 samples, respectively(Supplementary Table S1). The results showed that grade IIand grade III/IV cancer tissues possessed a higher level ofSPINDLIN1 expression than in grade I tissues (Fisher exacttest, P < 0.05). These findings corresponded with our firstreport, which identified SPINDLIN1 as a novel ovariancancer–related gene and showed that it was overexpressedin ovarian cancer tissues, but not in the normal tissues (3).

SPINDLIN1 promotes cancer cell proliferation andinvasionTo study the effects of SPINDLIN1 on cell proliferation,

6 independent clones, including 4 clones ofHeLa cells stablyexpressing SPINDLIN1 (SC1 to SC4) and 2 control clones(CC1 and CC2) were cultured (Fig. 2A). Three SPIN-DLIN1 clones and 2 control clones were used in colony-forming assays. After 2 to 3 weeks, the number of colonies ofHeLa cells increased from 129.33 � 24.01 (CC1) and111.33 � 7.64 (CC2) to 218.67 � 32.47 (SC1, N ¼ 3,P < 0.05), 358.00 � 44.58 (SC2, N ¼ 3, P < 0.001), and405.67� 45.08 (SC3,N¼ 3, P < 0.001; Fig. 2B), showingthat SPINDLIN1 promoted cell growth in vitro. To conductthe tumorigenicity assay, each of SPINDLIN1 clones (SC1,SC2, SC3, and SC4) were injected into one flank of 4 nu/nu

mice, respectively. CC1 and CC2 were injected into theother flank ofmice.Within 1month, it became clear that thetumors derived from SPINDLIN1-overexpressing HeLacells grew faster and were heavier than those produced bycontrol cells (Fig. 2C). The tumor weight increased from0.18� 0.13 g (CC) to 0.43� 0.28 g (SC,N¼ 16,P< 0.01).This result showed that SPINDLIN1 accelerated the growthof HeLa tumors. We used RNA interference to depleteSPINDLIN1 in HeLa cells (Supplementary Fig. S1A,siRNA-SPINDLIN1-1), leading to the slowed growth ofcells over 7 days in the CCK-8 assay (Supplementary Fig.S1B). Differences in proliferation appeared beginning onday 2 (N ¼ 6, P < 0.001). In addition, the colony-formingassays also confirmed that the depletion of SPINDLIN1resulted in reduced colony formation (N ¼ 3, P < 0.01;Supplementary Fig. S1C). These results suggested thatSPINDLIN1 depletion slowed the growth of tumor cells.An in vitro invasion assay showed a significant enhance-

ment of the invasiveness of HeLa and A549 cells transfectedwith SPINDLIN1 (Fig. 2D). The number of invasive HeLa

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Figure 1. The expression of SPINDLIN1 in cancer tissues. A, SPINDLIN1expression was analyzed by immunohistochemistry for samples on atissue microarray, including 53 cores of histologically confirmed ovariancancer samplesand14cores of normal tissues. SPINDLIN1expression invarious cancers (ii–iv) and normal ovarian tissues (i) are shown. Theindicated fieldwas enlarged. B, statistical data for the number of samplesexpressing and not expressing SPINDLIN1 from grade I to III/IV cancertissues are shown.Grade I,N¼6; grade II,N¼23; grade III/IV,N¼24. �,P< 0.05 compared with grade I (Fisher exact test).






cells increased from 88.27 � 4.20 to 262.33 � 13.70(N ¼ 3, P < 0.001) and that of A549 cells increased from114.73 � 12.63 to 322.67 � 18.77 (N ¼ 3, P < 0.001).These results showed that SPINDLIN1 contributed both tothe proliferation ability and invasiveness of the tumor cells.

SPINDLIN1 promotes activation of WNT/TCF-4signalingOur previous study suggested that SPINDLIN1 might

function as a tumor enhancer through activation of theWNT/TCF-4 signaling pathway (19). Here, we confirmed

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Figure 2. Effect of SPINDLIN1 protein on cancer cell proliferation and invasion. A, four clones of HeLa cells stably expressing pBPLV-SPINDLIN1 and2 clones of control cells were used in colony-forming assays. The expression of C-MYC-SPINDLIN1 in each clone was analyzed by Western blotting.B, five hundred cells per well were plated in 6-well plates and incubated for 2 to 3 weeks. Colonies were stained with crystal violet (left), and the numbers ofcolonies are shown (right). SC1, N ¼ 3, �, P < 0.05 compared with CC1 (Student t test). SC2, SC3, N ¼ 3, ��, P < 0.001 compared with CC1 (Student t test).SC, SPINDLIN1 clone; CC, control clone. C, a total of 3 � 106 HeLa cells expressing SPINDLIN1 (SC1–SC4) or control cells (CC1 and CC2) wereinjected into one flank of each of 4 nu/nu mice. One month after injection, the mice were sacrificed and tumors were removed (top left). The tumor weight(bottom left) and the tumor volume (right) are shown as the means � SD. N ¼ 16; ��, P < 0.01 compared with control (Student t test). D, HeLa andA549 cells overexpressing C-MYC-SPINDLIN1 or C-MYC (control) were used for the in vitro invasion assay. The cells that migrated through theMatrigel after36hourswere stainedwith crystal violet andare shownat randomly selected fields (left andmiddle). Thenumber of cells is expressedas themeans�SD (right).N ¼ 3; ��, P < 0.001 compared with control (Student t test).

Wang et al.





the direct interaction between SPINDLIN1 and TCF-4 byGST pull-down assay with b-catenin as a positive controland the GST empty vector as negative control (Fig. 3A).Next, we used a luciferase reporter assay to study the effectsof SPINDLIN1 on the activity of the TCF/b-catenin–responsive reporter. We found that SPINDLIN1 increasedthe activity of the pTOPFLASH reporter in both H1299cells (from 1.07 � 0.04-fold to 1.72 � 0.09-fold, N ¼ 3,P < 0.001) and A549 cells (from 1.05� 0.02-fold to 3.82�0.14-fold, N ¼ 3, P < 0.001). The activity of the controlreporter pFOPFLASH was set as 1.00-fold (Fig. 3B). Wethen confirmed the reduced activity of the TCF/b-catenin–responsive reporter by RNA interference against SPIN-DLIN1 (N ¼ 3, P < 0.01; Supplementary Fig. S1D). Totest whether SPINDLIN1 can affect the downstream targetsofWNT/TCF-4 signaling, the expression of C-MYC, cyclinD1, and Axin2 was analyzed by reverse transcription PCR(RT-PCR) and Western blotting. We found that theirexpression levels were substantially enhanced in A549 andH1299 cells transfected with SPINDLIN1 (Fig. 3C and D).These results suggested that SPINDLIN1 functions as aWNT/TCF-4 signaling pathway activator.

SPINDLIN1 promotes cancer cell proliferation viaWNT/TCF-4 activationTo address the question of whether SPINDLIN1 pro-

motes cancer cell proliferation in aWNT/TCF-4 activation-

dependent manner, we set out to analyze the biologicfunction of SPINDLIN1 mutants that have lost their abilityto activate the WNT/TCF-4 signaling pathway.According to our previous bioinformatics and localization

study, amino acid sites 84 and 99 were identified to playsignificant roles in the cellular localization of SPINDLIN1and may be involved in its function in the WNT/TCF-4signaling (19). First, the GST pull-down assay showed thatthe SPINDLIN1mutation at sites 84 and 99 (SPINDLIN1-84/99 M) decrease the interaction between SPINDLIN1and TCF-4 (Fig. 4A). The SPINDLIN1-84/99 M mutantwas observed to result in a sharp decrease in the TCF/b-catenin-responsive reporter's activity in comparison withwild-type SPINDLIN1 in the luciferase reporter assay (from5.60 � 0.62-fold to 1.15 � 0.01-fold, N ¼ 3, P < 0.001).The reporter's activity was almost as low as that for the emptyvector control (Fig. 4B). RT-PCR results showed a substan-tial decline inC-MYC, cyclinD1 andAxin2 expression levelswhen sites 84 and 99 of SPINDLIN1 were mutated (Fig.4C), and Western blotting showed that the mutationssignificantly decreased C-MYC and cyclin D1 protein levels(Fig. 4D). These data showed that mutation of SPINDLIN1at these sites abolished its function in the WNT/TCF-4signaling pathway.To study whether the mutations of SPINDLIN1 affects

its role in cancer cell proliferation, the SPINDLIN1double mutant was then transfected into cancer cells,

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Figure 3. The role of SPINDLIN1onWNT/TCF-4 signaling. A,GST-SPINDLIN1,GST-b-catenin (positive control), andGST (negative control) protein expressionwas induced in bacteria. Proteins were purified for GST pull-down and incubated with cellular lysates containing C-MYC-TCF-4. After separation by 10%SDS-PAGE, they were immunoblotted with anti-C-MYC antibody (input: 25 mg of protein extracts). B, in H1299 and A549 cells, pTOPFLASH wascotransfectedwith pcDNA3.1-MYC/HIS(�)-SPINDLIN1or pcDNA3.1-MYC/HIS(�), andpFOPFLASHcontrol vectorwas cotransfectedwith pcDNA3.1-MYC/HIS(�)-SPINDLIN1. The TCF/b-catenin–responsive activity was expressed in terms of relative luciferase activity, normalized to Renilla activity and isgiven as n-fold relative to the activity of control. Datawith error bars indicate themeans�SD.N¼ 3; ��,P < 0.001 comparedwith the level of OTþSPINDLIN1(Student t test). C and D, HeLa, A549, and H1299 cells were transiently transfected with pcDNA3.1-MYC/HIS(�)-SPINDLIN1 or pcDNA3.1-MYC/HIS(�)(control).mRNA (C) andprotein (D) levels ofC-MYC, cyclinD1, andAxin2were analyzedbyRT-PCRandWestern blotting, respectively.b-Actinwasusedas aninternal standard.






which was then used for colony-forming assays andtumorigenicity assays. As shown in Fig. 4E, the muta-tions caused a 43% reduction in the number of colonies(from 145.67� 6.03 to 82.67� 6.43, N¼ 3, P < 0.001)compared with wild-type, but the numbers were stillhigher than that observed in cells transfected with theempty vector. The tumorigenicity assay was also con-ducted for HeLa cells stably expressing the mutated or

wild-type SPINDLIN1. The tumor growth rate for themutant-transfected cells was markedly lower than for thewild-type SPINDLIN1, and the weight of mutants'tumors was reduced by 67% (from 0.35 � 0.11 g to0.11 � 0.14 g, N ¼ 6, P < 0.01; Fig. 4F). The resultssuggested that SPINDLIN1 promotes tumor cell prolif-eration by activating the WNT/TCF-4 signalingpathway.

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Figure 4. Mutation of SPINDLIN1 abolished its effects onWNT/TCF-4 signaling and cancer cell proliferation. A, GST, GST-SPINDLIN1, and GST-SPINDLIN1-84/99 M protein expression was induced in bacteria. Proteins were purified for GST pull-down (top) and incubated with cellular lysates containingC-MYC-TCF-4. After separation by 10% SDS-PAGE, they were immunoblotted with anti-C-MYC antibody (input: 25 mg of protein extracts, bottom).B, pEGFP-C1, pEGFP-C1-SPINDLIN1-84/99 M, and pEGFP-C1-SPINDLIN1-WT were used for luciferase reporter assays. The results were normalized toRenilla activity and are expressed as the ratio relative to the control. Data with error bars indicate the means � SD. N ¼ 3; ��, P < 0.001 compared with thewild-type (WT) level (Student t test). C andD, HeLa cells were transfectedwith plEGFP-C1, plEGFP-C1-SPINDLIN1-84/99M, or plEGFP-C1-SPINDLIN1-WT.RT-PCR (C) was carried out to analyze the mRNA levels of C-MYC, cyclin D1, and Axin2, and Western blot analyses (D) were conducted to determinetheir expression levels, using b-actin as an internal standard. E, HeLa cells transfected with GFP, GFP-SPINDLIN1-84/99 M, and GFP-SPINDLIN1-WT wereused for colony-forming assays (top left). Colonies were stained after 2 to 3 weeks (bottom left). The numbers are shown as the means � SD (right).��, P < 0.001 compared with wild-type level (Student t test). F, HeLa cells stably expressing SPINDLIN1-WT and SPINDLIN1-84/99 M were used fortumorigenicity assays. Each nu/nu mouse was injected with 3 � 106 cells in either flank. One month later, the mice were sacrificed and the tumors wereremoved (left). The tumor weight (middle) and the tumor volume (right) are expressed as themeans�SD.N¼ 6; ��,P < 0.01 compared with the wild-type level(Student t test).

Wang et al.





Aurora-A interacts with and phosphorylatesSPINDLIN1 at Ser84 and Ser99Considering the oncogenic characteristic of SPINDLIN1

in cancer cells and its key functional sites, Ser84 and Ser99were suggested to be candidate phosphorylation sites ofAurora-A by bioinformatics analysis. We hypothesized thatthe function of SPINDLIN1 may be related to Aurora-A, aSer/Thr kinase that plays a vital role in the initiation ofmitotic and G2–M transition and also takes part in manyactivities associated with carcinogenesis (20, 21). Aurora-A isfound at the centrosomes during interphase and at spindlepoles during mitosis (22, 23). We first checked the local-ization of SPINDLIN1 in GFP-SPINDLIN1–expressingHeLa cells, and endogenous Aurora-A was examined byimmunofluorescence with a TRITC-conjugated antibody.The results showed that SPINDLIN1 and Aurora-A local-ized approximately to the same region: they were both foundat the centrosome during interphase, at spindle poles frommetaphase to telophase, and at the microtubules of thecentral body during cytokinesis (Fig. 5A). The resultssuggested that the biologic functions of SPINDLIN1 andAurora-A might be related.To confirm the physical association between Aurora-A

and SPINDLIN1 in vitro, we conducted a GST pull-downassay (Fig. 5B). Coomassie-stained gels showed that recom-binant GST (used as a negative control) was expressed andpurified with the similar efficiency as the GST-SPINDLIN1fusion protein. Then, cellular lysates containing GFP-Auro-ra-A were incubated with purified GST-SPINDLIN1 orGST. Western blotting showed that GFP-tagged Aurora-Ainteracted with GST-SPINDLIN1, whereas band was notdetected in the sample incubated with GST alone. Theseresults showed their direct interaction in vitro.We used co-immunoprecipitation assays to investigate

whether Aurora-A interacts with SPINDLIN1 in vivo (Fig.5C). After cotransfection,HeLa cells with ectopic expressionof GFP-Aurora-A and C-MYC-SPINDLIN1 were lysed,and the lysates were immunoprecipitated using mouse anti-C-MYC antibody. The precipitates were analyzed by West-ern blotting. We observed an Aurora-A band, but this bandwas absent in the precipitates obtained using mouse IgG(control). These results showed that Aurora-A and SPIN-DLIN1 interacted in vivo.Subcellular colocalization results and the observed inter-

action between Aurora-A and SPINDLIN1 suggested thatSPINDLIN1 might serve as a substrate of Aurora-A kinase.To test this assumption, we conducted in vitro kinase assays.Recombinant Aurora-A and variousGST-fused fragments ofSPINDLIN1 were constructed as described above. Theresults of autoradiography showed that GST-SPINDLIN1is phosphorylated by Aurora-A, whereas GST alone was not(Fig. 5D, top). Meanwhile, we found that the kinasephosphorylated fragment 3 (amino acids 51–94) and frag-ment 6 (amino acids 85–138) of SPINDLIN1. We thennarrowed down the phosphorylation locations to aminoacids 61–94 and 85–105 (Fig. 5D, middle). To confirmthe specific phosphorylation sites, we replaced serines withalanines at S84, S85, S96, and S99 of SPINDLIN1 to

generate S-A mutants. The kinase assay showed a lack ofphosphorylation of the S84A and S99A mutants, showingthat these sites were the phosphorylation sites (Fig. 5D,bottom). The results showed that SPINDLIN1 interactswith Aurora-A, and its functional sites were phosphorylatedby this kinase.

DiscussionSPINDLIN1 was first identified as a new member of the

SPIN/SSTY gene family and was implicated in ovariancancers. Its expression is markedly upregulated in ovariancancer cells and other kinds of cancer cells.We have reportedthat the constitutive expression of SPINDLIN1 contributesto the malignant transformation of NIH3T3 cells (4). In ourprevious study, we showed that SPINDLIN1 is involved inmetaphase arrest and could affect chromosomal stability.SPINDLIN1protein is dynamically distributed alongmitot-ic spindle tubulins. We also showed that the overexpressionof SPINDLIN1 can induce cell-cycle delay inmetaphase andcause chromosomal instability (5). Nevertheless, the molec-ular mechanisms underlying the oncogenic role of SPIN-DLIN1 are still largely unknown.There seems to be a discrepancy in that SPINDLIN1

could cause a delay in mitosis and also cause chromosomeinstability (2, 5), but our data presented here confirm the factthat SPINDLIN1 stimulates cell growth. In fact, the 2phenomena are not completely contradictory. Indeed,cell-cycle delay might lead to defects in mitotic spindleorganization or DNA separation, ultimately resulting in cellapoptosis. However, it is possible that chromosomalcombination takes place in some cells overexpressing SPIN-DLIN1, which could help those cells escape cell apoptosisand obtain a selective growth advantage. Consequently,those cells undergoing apoptosis overcome the negativeinfluence of SPINDLIN1 and finally acquire an increasedgrowth rate. This possibility may be one explanation for whySPINDLIN1 overexpression induces tumorigenesis.Accumulating evidence has suggested that the WNT/

b-catenin signaling pathway plays an important role intumorigenesis and development (18, 24–26). Many fac-tors have been identified as cotranscription factors in thissignaling pathway. They are known to bind TCF-4 orb-catenin and function as transcription coactivators orinhibitors (27–29). In our previous study, we found thatSPINDLIN1 interacts with TCF-4 and activates its activ-ity (19), suggesting that SPINDLIN1 might promotecancer cell proliferation via this signaling pathway. Here,we have confirmed the existence of an interaction betweenthese 2 molecules in cancer cells. Ectopic expression ofSPINDLIN1 promotes the activity of a TCF/b-catenin–responsive reporter and enhances the expression levels ofC-MYC, cyclin D1, and Axin2, the targets of the WNT/TCF-4 signaling pathway. Amino acid sites 84 and 99 ofSPINDLIN1 were shown to be important for the nuclearlocalization of SPINDLIN1 in our previous study (19).The mutation of these 2 sites results in the abrogation ofthe nuclear localization of SPINDLIN1, which instead is






localized diffusely throughout the cell. Therefore, wehypothesized that the 2 sites could be important forSPINDLIN1 function. Here, we showed that amino acids84 and 99 of SPINDLIN1 are indispensable for thepromotion of WNT/TCF-4 signaling activity and cancercell proliferation. The results suggested that SPINDLIN1,with its functional sites at amino acids 84 and 99, func-tions as a cotranscription factor of the WNT/TCF-4signaling pathway, promoting cancer cell growth throughthe upregulation of C-MYC, cyclin D1, and Axin2expression.

It has been reported that ectopic expression of Aurora-A upregulates telomerase activity and increases C-MYCexpression in human ovarian and breast cancer cells(15, 30, 31). Knocking down C-MYC expression byRNA interference suppressed Aurora-A–stimulated telo-merase activity (32). Nevertheless, the mechanism bywhich Aurora-A upregulates C-MYC remains unclear. Inthis study, we have preliminarily discussed the possiblemechanism of Aurora-A. We found that SPINDLIN1interacts with Aurora-A and is a phosphorylated substrateof the kinase. In addition, the function of the

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Figure 5. Key functional sites of SPINDLIN1 could be phosphorylated by Aurora-A. A, HeLa cells grown on coverslips were transfected with GFP-SPINDLIN1and stained with Aurora-A antibody as well as DAPI. The different stages of the cell cycle from interphase to cytokinesis are shown. Scale bars, 10 mm.B, Coomassie-stained gels shows the prokaryotic expression and purification of recombinant GST and GST-SPINDLIN1 fusion proteins (top). TheWestern blotting shows the expression of GST-SPINDLIN1 and GFP-Aurora-A (middle). The proteins were mixed and incubated with glutathione beads,subjected to 10%SDS-PAGE, and immunoblotted (IB)with anti-Aurora-A antibody (input: 25mgof protein extracts, bottom). C,HeLa cells were cotransfectedwith GFP-Aurora-A and C-MYC-SPINDLIN1 (left). Total cell lysates (500 mg) were used for immunoprecipitation (IP) with anti-C-MYC antibody orcontrol mouse IgG. The immunocomplexes were analyzed by 10% SDS-PAGE and immunoblotted with anti-Aurora-A and anti-C-MYC antibodies (input: 25mg of protein extracts, right). D, the full-length SPINDLIN1 sample was divided into 5 aliquots, and the purified GST-SPINDLIN1 fragments and full-lengthprotein were incubated with immunoprecipitated Aurora-A in kinase buffer containing [g32-P]ATP. The samples were analyzed by 10% SDS-PAGE andautoradiographed to determine the Aurora-A–specific phosphorylation region of SPINDLIN1 (top). In the selected region, SPINDLIN1 fragments werefurther divided into 4 aliquots and used in the in vitro kinase assay to narrow down the phosphorylation region (middle). Mutants of SPINDLIN1-S84A,S85A, S96A, and S99A were used to identify the phosphorylated sites by in vitro kinase assay (bottom).

Wang et al.





phosphorylated sites of SPINDLIN1 has been shownhere. Therefore, we propose that the effects of Aurora-Aon tumorigenesis may be related to WNT/TCF-4 sig-naling, which is mediated by phosphorylation of thefunctional sites of SPINDLIN1. However, more researchwill be needed to investigate this complex process.

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

Grant SupportThe work was supported by The Major State Basic Research Program of China

(2009CB521704, 2011CB964804, 2011CBA01101), National Natural ScienceFoundation of China (30971145), and Beijing Natural Science Foundation(7092077).

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be herebymarked advertisement in accordance with18 U.S.C. Section 1734 solely to indicate this fact.

Received September 13, 2011; revised January 12, 2012; accepted January 12,2012; published OnlineFirst January 18, 2012.

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2012;10:326-335. Published OnlineFirst January 18, 2012.Mol Cancer Res Jing-Xue Wang, Quan Zeng, Lin Chen, et al. of WNT/TCF-4 SignalingSPINDLIN1 Promotes Cancer Cell Proliferation through Activation

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