KAI1 Overexpression Promotes Apoptosis and Inhibits Proliferation, Cell
Cycle, Migration, and Invasion in Nasopharyngeal Carcinoma cells
Zheng Guo, Yili Wang, Jing Yang, Jinghua Zhong, Xia Liu, Mingjun
Xu
PII:
DOI:
Reference:

S0196-0709(16)30208-3
doi: 10.1016/j.amjoto.2016.09.011
YAJOT 1751

To appear in:

American Journal of OtolaryngologyHead and Neck Medicine and Surgery

Received date:

7 August 2016

Please cite this article as: Guo Zheng, Wang Yili, Yang Jing, Zhong Jinghua,
Liu Xia, Xu Mingjun, KAI1 Overexpression Promotes Apoptosis and Inhibits Proliferation, Cell Cycle, Migration, and Invasion in Nasopharyngeal Carcinoma cells,
American Journal of OtolaryngologyHead and Neck Medicine and Surgery (2016), doi:
10.1016/j.amjoto.2016.09.011

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KAI1 Overexpression Promotes Apoptosis and Inhibits Proliferation, Cell Cycle,
Migration, and Invasion in Nasopharyngeal Carcinoma cells

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Zheng Guo, Yili Wang, Jing Yang, Jinghua Zhong, Xia Liu, Mingjun Xu*

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Department of Oncology, the first affiliated hospital of Gannan Medical University

*Correspondence: Mingjun Xu, Department of Oncology, the first affiliated hospital

China.
Email: guozheng39414@163.com

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Acknowledgements

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of Gannan Medical University, No. 23 of Qingnian Road, Ganzhou 341000, Jiangxi,

This study was supported by national natural science foundation of Jiangxi province

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(No 20142BAB205038), and science and technology research project of Jiangxi

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education department (No 14683).

Conflicts of Interest

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The authors declare no conflict of interests.

Abbreviations

NPC: nasopharyngeal carcinoma

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Abstract:
Objectives
The purpose of this study is to characterize the effect of KAI1 Overexpression on the

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biological behaviour of nasopharyngeal carcinoma (NPC) cells.

Background

Nasopharyngeal carcinoma is a highly malignant tumor with a high rate of incidence

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in China. Currently, there are no ideal therapeutic options for patients with NPC, but a
targeted therapy would have great potential for treating it. Therefore, there is an

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urgent need for novel therapeutic targets to provide new options for treating NPC. The
KAI1 gene was originally identified as a metastasis suppressor gene for advanced
human cancer. In NPC cell lines and tissues, the expression of KAI1 decreased as the
metastatic potential of cells increased, but its potential as a therapeutic target has not

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been elucidated.

Methods

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Non-transformed nasopharyngeal epithelium cell NP69 and NPC cell line C666-1
were cultured and KAI1 expression in these cells was detected by qRT-PCR and

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western blot. After the transfection of KAI1-pCDNA3.1 to NP69 and C666-1, the
KAI1 expression in these cells was detected by qRT-PCR and western blot, the
proliferation was performed by MTS, the cell cycle and apoptosis were performed by

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flow cytometry, the migration and invasion were examined by transwell.

Results

Our results showed that KAI1 was significantly upregulated in C666-1 cells compared
to that in NP69 cells. In addition, KAI1 overexpression significantly inhibited the
proliferation, cell cycle, migration, and invasion, and promoted apoptosis of C666-1
cells, but had no significant effect on NP69 cells.

Conclusion
Our findings suggest that KAI1 overexpression promotes apoptosis and inhibits
proliferation, cell cycle, migration, and invasion in NPC cells. We hypothesize that
KAI1 overexpression could be a potential therapeutic target for NPC.

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Keywords: nasopharyngeal carcinoma; KAI1, proliferation; cell cycle; apoptosis;

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migration; invasion

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1. Introduction
Nasopharyngeal carcinoma (NPC) is a highly malignant tumor arising from the
abnormal proliferation of epithelial cells lining the nasopharynx. There were an

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estimated 86,700 new cases and 50,800 deaths relating to NPC in 2012 worldwide[1].
NPC has a high incidence in select geographic and ethnic populations, such as
Southeast Asia and the southern parts of China, where over 33,000 new cases were

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diagnosed in 2012 [2]. In the southern parts of China, high incidence rates were
observed in the provinces including Guangdong and HongKong [3]. Currently, there

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are no ideal therapeutic options to treat patients with NPC, but a targeted therapy
would have a great potential to treat it. Therefore, there is an urgent need for novel
therapeutic targets to provide new options for the management of NPC.
The KAI1 gene, which maps to the human chromosome 11p11.2 and encodes a 267

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amino acid protein, belongs to the transmembrane 4 superfamily. The KAI1 gene was
originally identified as a metastasis suppressor gene for prostate cancer[4]. In

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advanced human cancer, the expression of KAI1 is generally downregulated, and is

positively associated with distant metastasis, lymph node metastasis,TNM stage, and

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poor prognosis[5-8]. The KAI1 promoter also responds to p53 and NM23-H1 [9]. In
pancreatic cancer, KAI1 overexpression decreased SRC and STAT3 phosphorylation,

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and inhibited expression of VEGF-C [10]. In human lung carcinoma cells, KAI1
overexpression promoted the expression of miR-203, downregulated expression of
FZD2, repressed the Wnt signaling pathway, and suppressed cancer metastasis [11].
In NPC cell lines, the expression of KAI1 decreased as the metastatic potential of
the cells increased. The expression of KAI1 in NPC tissues was significantly lower
than that in non-neoplastic nasopharyngeal tissues that is associated with lymph node
metastasis[12]. These results suggest that KAI1 can be a potential biomarker for poor
prognosis, but its potential as a therapeutic target has not been elucidated. In order to
study the therapeutic potential of KAI1 in NPC, we aimed to clarify the effect of
KAI1 on the biological behavior of NPC cells through analysis of KAI1 expression.
In this study, we found that KAI1 was significantly upregulated in C666-1 cells
compare to NP69 cells. In addition, KAI1 overexpression promoted apoptosis in

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C666-1 cells, but significantly inhibited proliferation, cell cycle, migration, and
invasion. However, no effects were observed in NP69 cells when KAI1 was

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overexpressed.

2. Materials and methods

2.1 Cell culture and plasmid transfection

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The NP69 (non-transformed nasopharyngeal epithelium cells derived from the

human nasopharynx) and C666-1 human NPC cell lines were kindly provided by

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Land Biology Co., Ltd (Guangzhou, Guangdong, China). Both cell lines were
cultured in RPMI-1640 media supplemented with 10% fetal bovine serum (FBS) and
100 U/ml penicillin/streptomycin (GIBCO BRL, NewYork, USA). All NPC cells were
cultured at 37C in a humidified incubator with 5% CO2. To overexpress KAI1, the

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open reading frame (ORF) of KAI1was synthesized by GENEWIZ (Suzhou, Jiangsu,

china) and linked to the pCDNA3.1 expression plasmid. When the NPC cells reached

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50% confluence, the cells were transfected with the plasmid using Lipofectamine
2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturers instructions.

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2.2 Quantitative real-time polymerase chain reaction (qRT-PCR)

Cells were harvested and total RNA was obtained from the cells using Trizol

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extraction (Invitrogen, Carlsbad, CA, USA). The total RNA was reverse transcribed
into cDNA using PrimeScript RT reagent kit with cDNA Eraser (Takara Bio, Dalian,
China) according to the manufacturers instructions. The qPCR was performed with
the SYBR Green master mix system (Applied Biosystem, Carlsbad, CA, USA). The
primers sequences for human KAI1: forward, 5-GCTCATGGGCTTCCTGGGCT-3
and reverse, 5-GAGCTCAGTCACGATGCCGC-3. Expression of GAPDH was used
as

an

internal

control.

The

ACACCCACTCCTCCACCTTT-3

primers
and

for

GAPDH:
reverse,

forward,

55-

TTACTCCTTGGAGGCCATGT-3. The qRT-PCR was performed on an Applied

Biosystems 7500 system (Applied Biosystems, Warrington, UK). Gene expression
was measured in triplicate, quantified using the 2CT method, and normalized to the
control [13].

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2.3 Western blot
Cells were harvested and lysed using RIPA buffer (Takara, Dalian, China). Total
protein concentration was determined using the BCA Protein Assay kit (Thermo

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Scientific, Rockford, IL, USA). Protein was separated on 12% SDS-PAGE and
transferred to PVDF membranes. The membranes were incubated with rabbit
anti-KAI1 (1:1000) or monoclonal rabbit anti--actin antibody (1:2000). Membranes

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were incubated with HRP-conjugated goat anti-rabbit IgG H&L secondary antibody
(1:10000) for 40 min and proteins were visualized using ECL (Thermo Scientific

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Pierce ECL Plus, Thermo Scientific, Rockford, IL, USA). The expression levels of the
proteins of interest were normalized against the expression level of -actin. All
antibodies were purchased from Abcam (Cambridge, MA, USA).
2.4 Cell proliferation assay

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Cell proliferation was evaluated using the CellTiter 96 AQueous One Solution Cell
Proliferation Assay kit (MTS) (Promega, Madison, WI, USA) according to the

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manufacturers instructions. Cells that were transfected with the KAI1 overexpression
plasmid or a control plasmidwere seeded into 96-well plates at a density of 5103 per

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well. After culturing for 0, 24, 48, and 72 h, 10 L of MTS was added to each well
and incubated at 37C for 4h. Absorbance at 490 nm was measured witha microplate

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reader (Thermo Scientific, Vantaa, Finland). The survival rate was calculated using
the proportion between the absorbance of KAI1-overexpressed and control cells.
2.5 Cell cycle and apoptosis analysis
At 48 h post transfection, each group of cells were digested with trypsin, washed
twice with PBS, and centrifuged at 2,000 g for 5 min to collect the cells. For cell
cycle analysis, the cells were fixed with pre-cooled 70% ethanol at 4C overnight,
digested with 200 g/ml ribonuclease A at 37C for 30 min, followed by the addition
of 100 l propidiumiodide (PI) at 4C in the dark for 30 min. For apoptosis analysis,
the cell pellet (1-5103 cells) was resuspended in 500 l binding buffer. Then, 5l
Annexin V-FITC and 5l PI was added and mixed at room temperature (protected
from light) for 15min. Apoptosis and the cell cycle were detected by flow cytometry
(BD Biosciences, San Jose, CA, USA). Each experiment was repeated three times.

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2.6 Transwell migration and invasion assays
Cell migration and invasion were assessed using a transwell migration assay. For
the migration assay, cells were harvested, re-suspended at a density of 5104 cells per

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200L in 0.1% serum medium, and were placed into the upper chamber of an insert
(8m pore size) (BD Biosciences, San Diego, CA, USA). The lower chamber was
filled with 10% FBS medium (600 L). The chamber was incubated at 37 oC and 5%

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CO2 for 48 h. For the invasion assay, 5103 cells were suspended in 250 l of
serum-free medium, seeded into the upper chamber that was pre-coated with 20 l of

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5 mg/ml Matrigel (BD Biosciences, San Diego, CA, USA), and incubated for 48 h.
After the removal of the cells from the upper chamber, the cells in the lower chamber
were fixed with 4% paraformaldehyde, stained with a 0.1% crystal violet solution in
20% ethanol for 2 h at room temperature. The migration and invasion cells were

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counted in five randomly selected fields using a LEICA microscope at 200

magnification and average counts were calculated. The assays were performed in

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triplicate.
2.7 Statistical Analysis

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The SPSS 19.0 software (IBM Inc., Chicago, IL, USA) was used to perform all
statistical analyses. Continuous variables are presented as mean standard deviation

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(SD). An independent sample t-test was used to compare the differences between
control and KAI1-overpression groups; P values <0.05 were considered statistically
significant.

3. Results
3.1 KAI1 is downregulated in the NPC cell lines
To examine endogenous KAI1 expression levels, qRT-PCR and western blot
analysis was performed on non-transfected NP69 and the C666-1 cells. The results
showed that KAI1 expression is decreased in the C666-1 cells compared to the NP69
cells (Figure 1).

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Figure 1 The expression of KAI1 in the NP69 and C666-1 cells.

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A: The expression of KAI1 in the NP69 and C666-1 cells was detected by qRT-PCR;
B: The expression of KAI1 in the NP69 and C666-1 cells was detected by western
blot. Data are presented as means SD. *P<0.05, vs. NP69 cell

3.2 Transfection with KAI1-plasmid could effectively increases KAI1 expression

levels
To investigate the role of KAI1 in NPC, we transfected the KAI1-pCDNA3.1
construct into NP69 and C666-1 cells. The expression level of KAI1 was then
examined by qRT-PCR and western blot. The results showed that KAI1 expression is
increased in both cell lines when transfected with the construct when compared to
control group (Figure 2).

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Figure 2 Transfection with KAI1-plasmid could effectively increases KAI1

expression level. A: The expression of KAI1 was detected by qRT-PCR; B: The

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expression of KAI1 was detected by western blot. Data are presented as means SD.
*P<0.05, vs. control group.

3.3 KAI1 overexpression suppresses proliferation of C666-1 cells

To determine whether KAI1 overexpression affects cell proliferation, a MTS assay
was performed. The results showed that KAI1 overexpression had no significantly
effect on the proliferation of NP69 cells at any time (Figure 3A), but significantly
suppressed the proliferation of C666-1 cells at 24h, 48h, and 72h compared to the
control group (Figure 3B).

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Figure 3 The effect of KAI1 overexpression on the proliferation of NP69 and C666-1
A: Overexpression of KAI1 did not significantly alter the survival ratio of NP69 cells.
B: Overexpression of KAI1 significantly suppresses the survival ratio of C666-1 cells.
Data are presented as means SD. *P<0.05, vs. control group.

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3.4 Overexpression of KAI1 suppresses cell cycle of C666-1 cells

To expose the underlying mechanism of suppression of cell proliferation by KAI1

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overexpression, stage distribution of the cell cycle was observed by flow cytometry.
These results are shown in Figure 4. Compared to the control group, overexpression

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of KAI1 induced a significant G1-phase arrest in C666-1 cells, significantly decreased

the percentage of cells in the S-phase, and prevented NPC cell replication. In contrast,

cells.

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overexpression of KAI1 had no effect on the cell cycle stage distribution in NP69

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Figure 4 The effect of KAI1 overexpression on the cell cycle of NP69 and C666-1
cells

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A: Representative images of the cell cycle of NP69 and C666-1 cells in the control
and KAI1-overpression group. B: Overexpression of KAI1 significantly altered the

means SD.

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cell cycle stage distribution in C666-1 cells but no effect on NP69. Data presented as

3.5 Overexpression of KAI1 promotes apoptosis in C666-1 cells

Flow cytometry was used to investigate the effects of KAI1 overexpression on
apoptosis. These results are shown in the Figure 5. Overexpression of KAI1 did not
significantly affect the percentage of apoptotic NP69 cells. In contrast, KAI1
overexpression significantly increased the percentage C666-1 cells undergoing
apoptosis.

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Figure 5 Effects of KAI1 overexpression on apoptosis in NP69 and C666-1 cells

A: Representative images of apoptotic NP69 and C666-1 cells in the control and

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KAI1-overpression group. B: Overexpression of KAI1 significantly altered the

percentage of C666-1 cells undergoing apoptosis but had no effect on NP69 cells.

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Data presented as means SD. *P<0.05, vs. control group.

3.6 Overexpression of KAI1 suppresses migration of C666-1 cells

To demonstrate the role of KAI1 in regulating C666-1 and NP69 cell migration, a
transwell assay was performed 48 h post transfection. The number of cells that passed
through the membrane onto the lower chamber was significantly decreased in the
KAI1-overpression group than in the control group in both NP69 and C666-1 cells
(Figure 6).

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Figure 6 The effect of KAI1 overexpression on the migration of NP69 and C666-1
A: Representative images of the migration of NP69 and C666-1 cells in control

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group and KAI1-overpression group. B: Overexpression of KAI1significantly

*P<0.05, vs. control group.

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inhibited the migration of C666-1 and NP69 cells. Data presented as means SD.

3.7 Overexpression of KAI1 suppresses invasion of C666-1 cells

To demonstrate the role of KAI1 in regulating C666-1 and NP69 cell invasion, a
transwell assay was performed 48 h post transfection. The ability of the cells to
invade through the membrane into the lower chamber was significantly decreased in
the KAI1-overpression group compared to that in the control group for C666-1 cells
(P<0.05) but had no effect on NP69 cells(Figure 7).

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Figure 7 The effect of KAI1 overexpression on the invasion potential of NP69 and
C666-1 cells

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A: Representative images of the invasion of NP69 and C666-1 cells in the control and
KAI1-overpression group. B: Overexpression of KAI1significantly decreased the

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number of invading C666-1 cells but had no effect on NP69 cells. Data presented as

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means SD. *P<0.05, vs. control group.

4. Discussion

The KAI1 protein was first identified as a metastasis suppressor in human cancer[4].
In the progression of malignancy tumors, KAI1 expression was significantly inhibited,
resulting in migrating and invading tumor cells [6, 7]. In this study, we found that the
expression of KAI1 was significantly upregulated in C666-1 cells compared to that in
NP69 cells. The C666-1 cell line is a NPC cell line having poorly differentiated and
high metastatic characteristics, whereas the NP69 cell line was derived from the
human nasopharynx epithelium. Our results showed that KAI1 expression was
downregulated in NPC cells. Previous studies have shown that in NPC tissue and cells,
KAI1 expression was decreased as the metastatic potential of cells increased, and is
associated with lymph node metastasis [12]. These previous results were consistent

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with our present results.
The biological behavior of KAI1 is closely related to tumor cells. Previous research
has shown that downregulation of KAI1not only promoted cell proliferation and

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inhibited apoptosis, but also promoted migration and invasion in human cancer,
leading to tumor progression and metastasis [14-16]. In this study, we found that
overexpression of KAI1 inhibited the survive rate and promoted apoptosis of NPC

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cells. Proliferation and apoptotic dysfunction plays an important role in NPC

tumorigenesis and progression. Inhibiting the proliferation and promoting apoptosis of

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NPC cells could suppress NPC progression. In addition, we found that overexpression
of KAI1 induced a significant G1-phase arrest in C666-1 cells, significantly decreased
the percentage of cells in the S-phase, prevented NPC cell replication, and reduced
cell proliferation. These results suggest that overexpression of KAI1 could prevent

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NPC cell replication, inhibit cell proliferation, and promote apoptosis. Furthermore,
we found that overexpression of KAI1 inhibited the migration and invasion of NPC

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cells. Cell migration and invasion is a central step in tumor metastasis.

Nasopharyngeal carcinoma is a highly metastatic tumor with a high mortality rate.

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Inhibiting NPC metastasis is the most effective way to treat NPC. Our results showed
that KAI1 has an important function on cell migration and invasion.

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Meanwhile, we studied the effect ofKAI1 expression on NP69 cells. We found that
overexpression of KAI1 did not affect the survival rate, the cell cycle, and apoptosis
in NP69 cells. In addition, we found that NP69 cells are unable to migrate and invade,
since overexpression of KAI1 did not affect the migration and invasion abilities. Our
results showed that overexpression of KAI1 did not damage the biological behavior of
normal nasopharyngeal epithelium cells.

5. conclusion
In a word, overexpression of KAI1 may help inhibit NPC cell replication,
proliferation, migration, and invasion, while promoting apoptosis without damaging
the biological behavior of normal nasopharyngeal epithelium cells. Our findings
hypothesize that KAI1 overexpression could be a potential therapeutic target for NPC.

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However, further research is needed to investigate the molecular mechanism of how
KAI1 affects the biological behavior of NPC cells and to verify our findings in animal

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models.

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