TIKI2 suppresses growth of osteosarcoma by targetingWnt/b-catenin pathway

Ruhui Li • Jianguo Liu • Hong Wu •

Lidi Liu • Lijun Wang • Shaokun Zhang

Received: 14 December 2013 / Accepted: 5 March 2014 / Published online: 27 April 2014

� Springer Science+Business Media New York 2014

Abstract Osteosarcoma is the most bone-associated

malignancy with high lethality. The current therapeutic

strategy benefits little on the survival of patients. Studies

have shown that aberrant activation of Wnt/b-catenin

pathway is essential for the progression of osteosarcoma,

implying that targeting this signaling may be an effective

way of therapeutics. Recently, TIKI family has been

identified as a new class of negative regulators for Wnt/b-

catenin pathway. However, the implication of TIKIs with

osteosarcoma has not been explored. Here, we constructed

an adenoviral vector that expresses TIKI2 in osteosarcoma

cells (Ad-TIKI2). TIKI2 expression was found to be

reduced in osteosarcoma specimens and cell lines. In tested

osteosarcoma cells, the activation of Wnt/b-catenin path-

way was found to be inhibited by TIKI2 expression. Fur-

thermore, the proliferation, colony formation ability, and

invasion were all significantly suppressed in osteosarcoma

cells infected with Ad-TIKI2. Finally, animal experiments

further confirmed that TIKI2 restoration was able to inhibit

the growth of osteosarcoma in vivo. Taken together, we

provided evidence that reduced expression of TIKI family

protein in osteosarcoma may participate in the progression

of osteosarcoma and restoring its expression was able to

impair the growth of osteosarcoma.

Keywords Osteosarcoma � TIKI � Wnt � b-Catenin

Introduction

As the most common bone cancers, osteosarcoma is fea-

tured with high invasiveness and metastasis as well as a

poor prognosis. Surgery is still the major modality for the

primary osteosarcoma. However, metastasis frequently

occurs in most cases. Chemotherapy is the only effective

way to treat osteosarcoma metastasis. But the patients still

have a poor survival after chemotherapy [1]. So far, the

molecular mechanisms of osteosarcoma progression and

metastasis have not been completely understood.

Wnt/b-catenin pathway plays an important role in the

initiation, progression, and metastasis of various cancers,

including osteosarcoma [2]. Several components of Wnt/b-

catenin pathway have also been identified as prognostic

biomarkers for osteosarcoma [3–5]. Suppressing Wnt/b-

catenin activation has also been shown to inhibit the pro-

liferation, tumorigenesis, and metastasis of osteosarcoma

cells [6–11]. The abnormal activation of Wnt/b-catenin

seemed to result from epigenetic alterations [12, 13].

Wnt family proteins, especially WNT5a, are secreted

from the osteosarcoma cells, and in turn, Wnts bind the

receptors located in the membranes of themselves and their

neighboring cells. These proteins can activate a down-

stream pathway that eventually leads to b-catenin stabil-

ization. Subsequently, b-catenin migrates into the nuclei

and acts as transcription factors in cooperation with TCF/

LEF to promote the expression of some oncogens, such as

c-myc and survivin [14, 15].

Ruhui Li and Jianguo Liu equally contribute to this work.

R. Li � L. Liu � L. Wang � S. Zhang (&)

Department of Spine Surgery, Norman Bethune First Hospital of

Jilin University, 71 Xinmin Street, Changchun 130021, China

e-mail: shaokunzhangcc@gmail.com

J. Liu

Department of Joint Surgery, Norman Bethune First Hospital of

Jilin University, Changchun 130021, China

H. Wu

Department of Ophthalmology, Norman Bethune Second

Hospital of Jilin University, Changchun 130041, China

123

Mol Cell Biochem (2014) 392:109–116

DOI 10.1007/s11010-014-2023-5

Many inhibitors of Wnt/b-catenin signaling have been

discovered, such as dickkopfs (DKK), Wnt inhibitory fac-

tors, and Secreted frizzled-related proteins [16]. Recently, a

new class of Wnt inhibitor, TIKI, has been identified to reply

on a novel mechanism to minimize Wnt-induced activation

of downstream pathways. Human has two TIKI orthologs,

TIKI-1 and TIKI-2. TIKIs consist of three functionally

different domains: signal peptide sequence, TIKI domain,

and transmembrane sequence. TIKI domain is revolution-

ally conserved from yeast to mammal. This protein harbors a

protease activity and removes several N-terminal amino

acids from the mature WNT proteins (no signal peptides).

Wnt proteins undergo consequent oligomerization and its

activity is highly compromised. Furthermore, silencing

TIKI2 expression can enhance the activation of Wnt/b-

catenin in HeLa and HEK-293 human cell lines [17]. This

new finding makes it a possibility that TIKI family may also

be implicated in the progression and initiation of osteosar-

coma. However, there is no report to address this issue.

In this study, we generated an adenoviral vector to

express TIKI2 to restore its expression in osteosarcoma

cells, and found that TIKI is able to suppress the activation

of Wnt/b-catenin pathway and growth of osteosarcoma

in vitro and in vivo.

Materials and methods

Cell cultures and compound

Human osteosarcoma cell lines, U2OS, SaOS2, KHOS, and

HOS, and human fibrablast cell lines, MRC-5, were pur-

chased from American Type Culture Collection (Manassas,

VA). Human normal liver cells L-02 were obtained from

Shanghai Cell Collection (Shanghai, China). Cells were

cultured using recommended media supplemented with

10 % of fetal bovine serum (FBS), 4 mM glutamine, 100

units/ml penicillin, and 100 lg/ml streptomycin in a 5 %

CO2 and humidified atmosphere at 37 �C.

BIO was purchased from Aldrich Sigma. 1 lM BIO was

added to the cell culture prior the subsequent experiments.

Osteosarcoma specimens

The osteosarcoma samples (n = 10) were obtained from

osteosarcoma patients by surgical resection according to

the procedures approved by Ethical Review Board in Jilin

University (Changchun, China).

Quantitative PCR (qPCR)

Total RNA was extracted from osteosarcoma and the cor-

responding noncancerous tissue as well as indicated cell

lines with Trizol solution (Sigma-Aldrich, MO), followed

by being transcribed into cDNAs using Rever Tra Ace

qPCR RT Kit (Toyobo, Japan) according to the manufac-

turer’s instructions. qPCR was performed using TaqMan�

2 9 Universal PCR Master Mix (Applied Biosystems) on

CFX96TM Real-Time PCR Detection System (Bio-Rad

Laboratories, CA) supplied with analytical software.

TIKI2-forward primer: 50-GACCTGCGTGCTGATC-30;TIKI2-reverse primer: 50-TAAAAGAAGATGACAG-30.Axin2-forward primer: 50-CCGGTGGACCAAGTCCT-

TAC-30; Axin2-reverse primer: 50-TCCATTGCAGGCAA

ACCAGA-30. Lgr5-forward primer: 50-TGAACACCTGC

TTGATGGCT-30; Lgr5-reverse primer: 50-TGCTGCGAT

GACCCCAATTA-30 [18].

Adenovirus construction

Ad-EGFP was kindly provided by Dr. Zhao (General

Hospital of Chengdu Military Area Command of Chinese

PLA, Chengdu, China) and used as control in our study.

Ad-TIKI2, which can express TIKI2 proteins, was con-

structed as follows. A TIKI2-encoding sequence was

obtained by PCR with total cDNA from HeLa cells as a

template. The used primers were as follows. Primer-for-

ward: 50-GCCGTCGACACCATGCACGCCGCCCTG-30;Primer-reverse: 50-GCCGATATCTCAGGAGGGCCCAA-

30. The product was digested with SalI and EcoRV, and

then, inserted into pShuttle-CMV, generating pShuttle-

CMV-TIKI2. Subsequently, pShuttle-CMV-TIKI2 and

pAdEasy were cotransfected into HEK-293 cells. After

plague purification and PCR-based identification, the

recombinant adenoviruses were harvested and then purified

with the CsCl gradient centrifugation, namely Ad-TIKI2.

The involved adenoviruses were titrated with TCID50

method on HEK-293 cells and shown as plaque-forming

units per milliliter (pfu/ml). The structures of these ade-

noviruses are shown in Fig. 2a.

Immunoblotting assay

To examine the expression level of phosphorylated and

total b-catenin, cells were infected with the indicated

adenoviruses of 10 MOI. After 48 h, proteins were har-

vested with M-PER� Mammalian Protein Extraction

Reagent (Thermo Scientific, IL), separated by polyacryl-

amide gel electrophoresis and transferred onto 0.45 lm

nitrocellulose membranes. After 2 h blocking with 5 % fat-

free dry milk, the membrane was then incubated with

primary antibodies for 2 h. The membrane was incubated

with corresponding secondary antibody for 1 h, and finally,

visualized with SuperSignal West Dura Extended Duration

Substrate (Thermo Scientific, IL).

110 Mol Cell Biochem (2014) 392:109–116

123

TCF reporter assay

The activation of Wnt signaling was further examined by

TCF reporter dual-luciferase assay. Cells were seeded in

24-well tissue culture plates and transfected with TOPflash

vectors (Millipore, MA) and renilla luciferase reporter

vector (pRL-TK) (40:1) (Promega, WI) with Lipofect-

amine 2000 (Invitrogen). Overnight, the tested cells were

infected with the indicated adenoviruses of 10 MOI. 48 h

later, cells were harvested using lysis buffer (Promega, WI)

and luciferase activities were monitored for luciferase and

renilla activity using reagents in dual-luciferase reporter

assay kit and a luminometer (Promega), as recommended

by the manufacturer. All experiments were performed for

three times.

MTT assay

Adenoviruses of indicated MOIs were added to cell cul-

tures. 48 h later, 50 ml 3-(4,5-dimethylthiazol-2-yl)-2,5-

diphenyltetrazolium bromide (MTT) (1 mg/ml) was added.

4 h later, MTT was removed and 150 ml dimethyl sulf-

oxide was added. The spectrophotometric absorbance was

measured on a model 550 microplate reader (Bio-Rad

Laboratories, Hercules, CA, USA) at 570 nm with a ref-

erence wavelength of 655 nm. Cell viability was calculated

according to the following formula: Cell viabil-

ity = absorbance value of infected cells/absorbance value

of uninfected control cells.

Cytometrical analysis of apoptotic rates

The cells were transduced with indicated adenoviruses of

ten MOI for 48 h. Then, the cells were harvested, fixed in

70 % ethanol and stained with Propidium Iodide (PI,

200 mg/ml) for cytometrical analysis with Aria II sorter

(BD Biosciences). For each group, 10,000 cells were

counted to determine the percentages of sub-G0/G1

population.

Contact-independent colony formation assay

To determine contact-independent colony formation effi-

ciency, cells were plated into low-attachment 96-well

plates (Costar) at ten cells per well. The cells were infected

with the indicated adenoviruses of ten MOI and maintained

in stem cell medium. Equal volume of fresh medium was

replaced to replenish growth factors and nutrients every

3 days. Methyl cellulose (1 %) (Sigma Aldrich) was added

to prevent cell aggregation. Cells were incubated for

14 days and then the number of wells which contained

spheres with a diameter higher than 75 lm was counted.

The experiments were performed for three times.

Transwell assay

The invasiveness of osteosarcoma cells was evaluated by

transwell assay in a 24-well plate (Corning, Cambridge,

USA) after the indicated adenoviruses were added. The

transwell membrane with 8.0 lm pore was coated without

or with Matrigel (1 lg/ll) (BD Biosciences, San Diego,

USA). Cells was infected with indicated adenoviruses of

ten MOI, suspended in DMEM/F12 basic medium, and

seeded in the upper chambers (5 9 104 cells each). The

nutritional attractant in the lower chambers was DMEM/

F12 medium containing 10 % FBS. The cells were then

allowed to migrate for 24 h. Then, the migrant cells on

membranes of the lower chambers were stained with

crystal violet and counted in ten randomly selected fields

(1009). The cells in at least ten randomly selected

microscopic fields at 1009 magnification were counted and

expressed as invasive cells per microscopic field. All the

experiments were performed for three times.

Animal experiments

Procedures for animal experiments were all approved by

the Committee on the Use and Care on Animals in Jilin

University (Changchun, China).

KHOS tumor xenograft was established by injecting

2 9 106 cells at the right flanks of 5-week-old male BALB/

c nude mice. As soon as tumors grew to 6–8 mm in

diameter, 18 mice were randomly and equally divided into

three groups (n = 6). The mice were intratumorally

injected with PBS, Ad-EGFP (5 9 108 pfu), or Ad-TIKI2

(5 9 108 pfu), respectively.

Tumor diameter was measured by periodic measure-

ments with calipers and volume was calculated using the

following formula: tumor volume (mm3) = maximal

length (mm) 9 perpendicular width (mm)2/2. The tumor

weights were measured when the mice were sacrificed.

Results

TIKI2 expression was reduced in osteosarcoma

specimens

The expression profile of TIKI2 has been explored in

osteosarcoma, so we examined the expression levels of

TIKI2 proteins in osteosarcoma. qPCR assays were per-

formed to determine the abundance of TIKI2 mRNA in the

tested osteosarcoma specimen. The data revealed that

TIKI2 mRNA was underexpressed in osteosarcoma sam-

ples (n = 10), in comparison with the corresponding non-

cancer tissue (Fig. 1a). Consistently, TIKI2 mRNA was

Mol Cell Biochem (2014) 392:109–116 111

123

B

Rel

ativ

eTIK

I2 m

RN

A le

vel

0

0.5

1

1.5

2

MRC-5L-0

2

U2OS

SaOS2

KHOSHOS

Rel

ativ

eTIK

I2 m

RN

A le

vel

A

0

0.5

1

1.5

2

** ****

*

*

Fig. 1 The abundance of TIKI2 was reduced in osteosarcoma

specimen and cell lines. a TIKI2 expression level was quantified in

osteosarcoma specimen and their corresponding noncancerous tissues

by qPCR assays (n = 10). The lines represented the average values of

each group. b TIKI2 mRNA expression was detected in osteosarcoma

(U2OS, SaOS2, KHOS, and HOS) and normal cell lines (MRC-5 and

L-02) by qPCR assays. The relative values were shown as

mean ± SD

A

Ad-EGFP

Ad-TIKI2

B

p-β-catenin

GAPDH

Nuclear β-catenin

U2OS KHOS

C

0

0.2

0.4

0.6

0.8

1

1.2

1.4 Ad-EGFPAd-TIKI2

Rel

ativ

e TO

Pfl

ash

/Ren

illa

D

0

0.2

0.4

0.6

0.8

1

1.2

1.4

U2OS KHOS U2OS KHOS U2OS KHOS

Ad-EGFP

Ad-TIKI2

Rel

ativ

e m

RN

A le

vels

Axin2 Lgr5

**

*** **

***

Fig. 2 TIKI2 expression

suppressed the activation of

WNT pathway in osteosarcoma

cells. a The structure of

adenoviruses expressing TIKI2,

Ad-TIKI2, was illustrated as

well as control vector, Ad-

EGFP. b The expression level of

phosphorylated and nuclear

b-catenin was determined in

osteosarcoma cells, U2OS and

KHOS, infected with Ad-EGFP

and Ad-TIKI2 by immunoblot

assays. GAPDH was used as

endogenous reference. c The

transcription activity of TCF/

LEF was detected in U2OS and

KHOS cells under the above

treatments using TOPFlash

reporter assays. The bars

represented mean ± SD of

three independent experiments.

d The mRNA level of WNT

target genes, Axin2 and Lgr5,

was assessed in osteosarcoma

cells after the transduction of

indicated adenoviral vectors.

The bars represented

mean ± SD of three

independent experiments

112 Mol Cell Biochem (2014) 392:109–116

123

also found to be reduced in the tested osteosarcoma cell

lines (Fig. 1b).

Adenovirus-mediated TIKI2 expression suppresses

the activation of WNT pathway in osteosarcoma cells

Subsequently, we employed an adenoviral vector that

expresses TIKI2 (Ad-TIKI2) to detect if TIKI2 overex-

pression was able to affect the activation of Wnt/b-catenin

pathway (Fig. 2a). Immunoblot analysis of phosphorylated

and nuclear b-catenin revealed that the activation of Wnt/

b-catenin signaling was greatly suppressed under the

infection of Ad-TIKI2 (Fig. 2b). Also, the activity of

TOPflash reporter was also determined in the cells infected

with Ad-EGFP and Ad-TIKI2. The results indicated that

Wnt/b-catenin activation was greatly reduced in osteosar-

coma cell lines when TIKI2 expression was restored

(Fig. 2c). Finally, we detected if TIKI2 expression can

affect the transcription of target genes, Axin2 and Lgr5,

downstream of Wnt/b-catenin signaling (Fig. 2d).

TIKI2 restoration compromises the proliferation,

survival, invasiveness, and colony formation

of osteosarcoma cells

Subsequently, we studied the effect of TIKI2 restoration on

the behaviors of osteosarcoma cells. MTT assays showed

that Ad-TIKI2 was able to reduce the proliferation rates of

osteosarcoma cells (Fig. 3a). The detection of sub-G0/G1

subpopulation revealed that TIKI2 expression increased the

percentage of apoptotic osteosarcoma cells (Fig. 3b).

Invasion capability of U2OS and KHOS cells was also

found to be suppressed under the infection of Ad-TIKI2

(Fig. 3c). In addition, soft agar assays were used to

examine the effect of TIKI2 expression on the colony

formation ability of osteosarcoma cells (Fig. 3d).

The growth of osteosarcoma xenograft was inhibited

by TIKI2 expression in mice

The effect of TIKI2 overexpression was further investi-

gated in vivo. KHOS cells were subcutaneously injected

Nu

mb

er o

f sp

her

oid

co

lon

y-co

nta

inin

g w

ells

Inva

sive

cel

ls

Su

b-G

0/G

1ce

lls (

%)

C D

B

U2OS KHOS

Pro

lifer

atio

n f

old

s

Time (h)

A

0%

10%

20%

30%Ad-EGFPAd-TIKI2

1

1.5

2

2.5

3

3.5

4 Ad-EGFP

Ad-TIKI2

1

1.5

2

2.5

3

3.5

4 Ad-EGFP

Ad-TIKI2

0

20

40

60

80

100

U2OS KHOS24 48 72 96 24 48 72 96

U2OS KHOS

Ad-EGFP

Ad-TIKI2

*

**

****

*

****

**

***

Ad-EGFP

Ad-TIKI2

Ad-EGFP

Ad-TIKI2

U2OS KHOS

Fig. 3 TIKI2 expression mediated by adenovirus exerted anti-tumor

activity on osteosarcoma cells. a The proliferation rates of osteosar-

coma cells infected with Ad-EGFP or Ad-TIKI2 were detected with

MTT assays at the indicated time points. b The sub-G0/G1 population

was quantified in Ad-EGFP- or Ad-TIKI2-transduced osteosarcoma

cells using flow cytometry. The bars represented mean ± SD of three

independent experiments. c The invasive cells of osteosarcoma cells

overexpressing EGFP or TIKI2 were counted with transwell assays.

The bars represented mean ± SD of three independent experiments.

The representative images were shown (9200). d The colony

numbers of osteosarcoma cells were determined under the infection

of adenoviral vectors expressing EGFP or TIKI2. The bars repre-

sented mean ± SD of three independent experiments

Mol Cell Biochem (2014) 392:109–116 113

123

into the flanks of mice to establish an osteosarcoma

xenograft model. PBS, Ad-EGFP, and Ad-TIKI2 were in-

tratumorally injected, followed by periodically measuring

the diameter of these tumors. Their volumes were calcu-

lated and shown in Fig. 4a. The tumors were also weighted

after sacrificing these mice (Fig. 4b, c). The data showed

that TIKI2 expression was able to suppress the growth of

KHOS osteosarcoma xenograft in mice.

The suppression of WNT pathway is required

for the inhibitory effect of TIKI2 on osteosarcoma cells

To confirm the requirement of WNT pathway deactivation

for the role of TIKI2 in anti-tumor activity on osteosar-

coma cells, we employed BIO, a small molecule WNT

activator, to prevent WNT pathway from suppression

induced by TIKI2. MTT assays revealed that the prolifer-

ation rates were increased in U-87 MG co-treated with Ad-

TIKI2 and BIO, compared with that infected with Ad-

TIKI2 alone (Fig. 5a). The flow cytometrical analysis of

sub-G0/G1 population also indicated that the percentages of

apoptotic cells were reduced in U-87 MG co-treated with

Ad-TIKI2 and BIO, in comparison with that expressed

TIKI2 (Fig. 5b). Transwell assays showed that BIO

restored the invasiveness of U-87 MG cells treated with

Ad-TIKI2 (Fig. 5c). Furthermore, there was no significant

difference in the colony formation ability between osteo-

sarcoma cells, and Ad-EGFP. In contrast, the number is

higher in cells co-treated with Ad-TIKI2 and BIO than Ad-

TIKI2 alone (Fig. 5d).

Discussion

In this study, we investigated if TIKI2, a protease for WNT

degradation, was implicated with osteosarcoma and its

activation of Wnt/b-catenin signaling. Subsequent experi-

mental data revealed that TIKI2 was downregulated in

osteosarcoma and may activate Wnt/b-catenin pathway in

osteosarcoma cells. This is the first time to provide evi-

dences that TIKI family is associated with the biological

traits of cancer cells.

Numerous evidences have demonstrated that aberrant

Wnt/b-catenin signaling has a key role in the progression

of osteosarcoma [19]. The proliferation, survival, and

invasion have been shown to be affected by Wnt/b-catenin

signaling. So far, some miRNAs and proteins have been

found to suppress the activation of WNT/b-catenin path-

way and have anti-osteosarcoma activity [13, 20]. Target-

ing Wnt/b-catenin pathway should be an effective way to

treat osteosarcoma. b-catenin siRNA has been found to

suppress the activation of Wnt/b-catenin signaling [21].

However, its outcome is not desirable due to inefficient

transfection.

Osteosarcoma gene therapy has been studied for dec-

ades. The selected tumor suppressor genes are very

important for the treatment. To date, some genes have been

tested for osteosarcoma therapy. For example, TRAIL has

been verified as an effective candidate to treat bone cancers

with adenoviruses as vectors [22]. In our study, TIKI2 has

been shown to be effective to suppress the proliferation,

survival, invasiveness, and tumorigenesis of osteosarcoma

0

500

1000

1500

2000

2500

3000

0 3 7 14 21 28 35 42

PBS

Ad-EGFP

Ad-TIKI2

0

20

40

60

80

100

120

140

160BA

Tum

or

size

(m

m3 )

Tum

or

wei

gh

t (m

g)

Time (days)

**

*

*C

PBS

Ad-EGFP

Ad-TIKI2

Fig. 4 Ad-TIKI2 reduced the growth of KHOS osteosarcoma xeno-

graft. a Tumor sizes were measured after the intratumoral injection of

PBS, Ad-EGFP, and Ad-TIKI2 (5 9 108 pfu) (n = 6). The dots

represented mean ± SD of all individuals in each group. b The

tumors were weighted after sacrificing. The dots presented individual

mice with lines as average values. c Some representative images of

tumors injected with PBS, Ad-EGFP, or Ad-TIKI2 were shown

114 Mol Cell Biochem (2014) 392:109–116

123

cells. Therefore, TIKI2 can also be used for osteosarcoma

gene therapy in future.

In future, TIKI can be further studied for better under-

standing this promising WNT inhibitor. For example, what

transcriptional factors drive the expression of TIKI family?

Is there post-translational mechanism to regulate TIKI

family? Can TIKI family affect more pathways other than

WNT? It is believed that more knowledge on TIKI family

can contribute to better understanding of WNT pathway.

Taken together, we found that TIKI family is underex-

pressed in osteosarcoma and its downregulation may

account for the activation of Wnt/b-catenin pathway in this

malignant disease. Restoring the expression of TIKI family

proteins may be a promising strategy to treat

osteosarcoma.

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bars represented mean ± SD of three independent experiments

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