Oral Oncology 50 (2014) 983990

Contents lists available at ScienceDirect

Oral Oncology
journal homepage: www.elsevier.com/locate/oraloncology

Beclin1 inhibits proliferation, migration and invasion in tongue

squamous cell carcinoma cell lines
Junquan Weng 1, Cheng Wang 1, Yawen Wang, Haikuo Tang, Jianfeng Liang, Xiqiang Liu,
Hongzhang Huang, Jinsong Hou
Department of Oral and Maxillofacial Surgery, Guanghua School of Stomatology, Hospital of Stomatology, Sun Yat-sen University, Guangzhou, Guangdong 510055, China
Guangdong Provincial Key Laboratory of Stomatology, Sun Yat-sen University, Guangzhou, Guangdong 510055, China

a r t i c l e

i n f o

Article history:
Received 26 January 2014
Received in revised form 21 June 2014
Accepted 30 June 2014
Available online 2 August 2014
Keywords:
Beclin1
Autophagy
Proliferation
Invasion
Migration
Tongue squamous cell carcinoma

s u m m a r y
Objectives: The role of autophagy is still a controversy in cancer development. In our previous study, we
conrmed that decrease of autophagy activity promotes malignant progression of tongue squamous cell
carcinoma (TSCC). However, the role of autophagy-related protein, Beclin1, has not well been documented in TSCC. In this study, we aim to elucidate the role of beclin1 in TSCC progression and investigate
its potential mechanisms.
Materials and methods: TSCC cell lines, SCC9 and SCC15 were used to generate the stable cells with transfection lentivirus BECN1 and sh-BECN1. Then, Beclin1 expression was detected with qPCR and western
blot. Moreover, the expressions of autophagy-related proteins and tumor metastasis associated proteins
were examined by western blot and ELISA. For functional analysis, MTT assay were performed to evaluate
the proliferation activity and transwell assay was used to assess the migration and invasion ability.
Finally, TSCC xenograft models were established to conrm the effect of Beclin1 on TSCC in vivo.
Results: The results showed that BECN1 and sh-BECN1 virus transfection signicantly increased or
decreased the mRNA and protein expression of Beclin1 in the transfected TSCC cells. Meanwhile, we also
observed that Beclin1 could enhance the expression levels of LC3-II, ATG4 and ATG5. Then, we revealed
that overexpression of Beclin1 inhibited proliferation, migration and invasion while knockdown of
Beclin1 promoted proliferation, migration and invasion in TSCC cells. Furthermore, we demonstrated that
vascular endothelial growth factor (VEGF), matrix metalloproteinase-2 and -9 were involved in Beclin1mediated inhibition of migration and invasion. More importantly, our data also conrmed that Beclin1
inhibited TSCC xenograft growth in vivo.
Conclusion: Taken together, the results indicate that autophagy regulating gene, Beclin1, may contribute
to the malignant phenotypes of TSCC cells and can be a potential target for oral cancer gene therapy.
2014 Elsevier Ltd. All rights reserved.

Introduction
Tongue squamous cell carcinoma (TSCC) is the most common
cancers of oral cavity, which demonstrates much more aggressive
behavior [1,2]. Despite improvements in treatment, the survival
of patients with TSCC has not been signicantly improved over
the past several decades. Local or regional relapse and cervical
lymph node metastasis are the most prevalent causes of death in

Corresponding author at: Department of Oral and Maxillofacial Surgery,

Guanghua School of Stomatology, Hospital of Stomatology, Sun Yat-sen University,
Guangzhou, Guangdong 510055, China. Tel.: +86 2083862531; fax: +86
2083822807.
E-mail address: houjsgz01@yahoo.com (J. Hou).
1
These authors contributed equally to this study.
http://dx.doi.org/10.1016/j.oraloncology.2014.06.020
1368-8375/ 2014 Elsevier Ltd. All rights reserved.

these patients [3,4]. So, many studies have been performed to

investigate the effects of various biological factors on the aggressive potentials of TSCC and unravel the molecular mechanism of
TSCC pathogenesis [57]. However, these processes are still poorly
understood. Autophagy is an orchestrated intracellular process in
which cytoplasmic constituents are transported by autophagosomes to lysosomes for degradation, thus is essential for growth
regulation, cell differentiation and cellular homeostasis [810].
The mammalian autophagy gene Beclin1, is a coiled-coil protein
involved in the localization of autophagic proteins to a pre-autophagosomal structure [1113]. In addition, Beclin1 regulates
autophagy at different steps by binding to different Beclin1-interacting proteins [1416]. Beclin1 and its associated proteins may
also have anti-tumor activity, potentially by modulating autophagic cell death [17,18]. Haploid deletion of Beclin1 is frequently

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J. Weng et al. / Oral Oncology 50 (2014) 983990

detected in human breast and ovarian cancers [11,19]. But, the role
of Beclin1 is still not clear in the progression of TSCC. In our previous study, we conrmed that down-regulation of Beclin1 is a frequent event in TSCC, and its decreased expression was associated
with T stage, clinical stage and differentiation [20]. In this study,
we further elucidated the role of Beclin1 and then investigated
its potential mechanisms in TSCC progression.

Materials and methods

Cell lines and cell culture
Human tongue squamous cell carcinoma cell lines (SCC-9 and
SCC-15) were maintained in Dulbeccos modied Eagles medium
(DMEM, Gibco) containing 10% fetal bovine serum (Gibco), penicillin (100 units/ml), and streptomycin (100 lg/ml) at 37 C with 5%
CO2 in a humidied air atmosphere containing.
Plasmids and transfection
Lentiviral shRNAs were used to knockdown Beclin1 expression
in SCC-9 and SCC-15 cells. Cells were incubated with lentivirus
particles and polybrene 8 lg/ml for 16 h and washed with medium. Then, cells were selected with 0.5 lg/ml puromycin for
2 weeks and qPCR and western-blot were performed to conrm
the expression of Beclin1. Five different oligonucleotide sequences
of Beclin1 shRNA were tested for optimal knockdown of genes. The
selected sequences and negative control sequences were shown in
Supplementary Table 1. For overexpression of Beclin1, the human
Beclin1 cDNA (Open Biosystems, USA) was PCR amplied and
cloned into the lentivirus vector with the CMV promoter. Then,
cells were transfected with lentivirus with our without Beclin1
cDNA followed by selection with puromycin (0.5 lg/ml). Western-blot and qPCR were performed to validate its expression.

Enzyme linked immunosorbent assay

To further quantify and conrm the effects of Beclin1 on VEGF
and gelatinolytic MMPs observed by western blotting, we carried
out an enzyme linked immunosorbent assay. Cells were seeded
on 24-well plates to grow to sub-conuent. Then, the cells were
washed with PBS and incubated with serum-free medium for
48 h. The culture medium were collected, briey centrifuged and
stored at 20 C. VEGF, MMP-2 and MMP-9 concentrations in the
medium were analyzed by commercial enzyme-linked immunosorbent assay (ELISA) kits (Cell Signaling, USA) according to the
manufacturers instructions.
Cell proliferation analysis
Cells were seeded in a 96-well plate at a density of 1  104/well
followed by incubation at 37 C for 24 h. Then, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (Sigma, USA) was added into
each well according to the instructions of the manufacturer and
subsequently dimethyl sulfoxide (Sigma, USA) was added followed
by vortexing gently for 15 min. Absorbance was determined at
492 nm with a microplate reader. Cells from each group were
added to 5 wells and experiment was performed in triplicate.
Absorbance was detected at 2496 h after transfection, and growth
curve was delineated with absorbance as the vertical axis and time
as the horizontal axis.
Cell migration and invasion assay
The cell migration and invasion were measured using the BD
BioCoat system (BD Biosciences, USA) following the manufacturers
instructions. In brief, cells were seeded in the upper chambers, and
culture medium with 10% FBS was added to the lower chambers.
For invasion assay, inserts coated with matrigel were used. After
24 h incubation, cells which migrated to the reverse side of inserts
were stained with DAPI and quantied.

Real time PCR analysis

Establishment of sub-cutaneous xenograft tumor model in nude mice

Total RNA was isolated with the RNeasy Total RNA kit (Invitrogen, USA). cDNA was generated with the qScript cDNA synthesis kit
(Roche, Germany) according to the instructions of the manufacturer. Quantitative gene expression was performed for Beclin1,
VEGF, MMP2 and MMP9 using LightCycler 480 SYBR Green I Master Mix Reagent Kit (Roche, Germany) and the LightCycler 480
Real-time System (Roche, Germany). The data were normalized
to the internal control, GAPDH to obtain DCt. The nal amount of
gene of interest relative to control samples was reported by
2 DDCt method.

Twenty-ve male BALB/c nude mice (male, 68-week-old), randomly divided into ve groups, were housed in a temperature-controlled, pathogen-free animal facility with 12-h light and dark
cycles. Approximately 107 of lentivirus-infected, mock-infected
and parental SCC-9 cells in 200 ll of sterile PBS were injected subcutaneously into the dorsal region to establish tumors. Tumor
mass (xenograft) volume was measured every week from week 1
to week 5. After 7 week, mice were sacriced by cervical dislocation, and tumors were harvested. The research was approved by
the Ethical Committee on Animal Research of the Sun Yat-sen
University. All experimental procedures were performed according
to national guidelines regarding the care and use of laboratory
animals.

Western blot analysis

Cellular protein were collected and lysed with RIPA buffer with
PMSF (Beyotime Biotechnology, China). The lysates were centrifuged at 15,000g for 10 min at 4 C. Further, to analyze the secretion of VEGF, MMP-2, and MMP-9 in culture supernatants, cells
were incubated in serum-free medium for 24 h and then collected
and concentrated by a speed vacuum. Then, the samples were separated by SDSPAGE and transferred to the PVDF membranes. After
probing with primary antibodies overnight at 4 C and secondary
antibodies for 1 h at room temperature, the bands were detected
by enhanced chemiluminescence (ECL). Primary antibodies used
are as follows: Beclin1, VEGF, IgG (Santa Cruz, USA); LC3, MMP-2,
MMP-9 (Cell Signaling, USA), GAPDH (Beyotime Biotechnology,
China).

Immunohistochemistry
Immunohistochemical studies on the xenograft tissue microarrays were performed according to our previous protocol [20].
Beclin1, ATG5, VEGF, MMP2 and MMP9 staining intensity were
graded into three categories based on the percentage of positive
TSCC cells: weak, 535%; moderate: 3570%; strong, >70%.
Statistical analysis
Data were analyzed using the Statistical Package for the Social
Science (SPSS, Chicago, USA), Version 17.0. All values are presented
as mean standard deviation (SD). The Student t test was used for

J. Weng et al. / Oral Oncology 50 (2014) 983990

paired data that were normally distributed. A p-value <0.05 was

considered statistically signicant.
Results
Autophagy-related proteins expression in TSCC cells with or without
LV-Beclin1 and LV-shBec treatment
To investigate the role of Beclin1 on autophagy, growth, migration and invasion in SCC-9 and SCC-15 cells, we rst established
two model systems in vitro in which Beclin1 was overexpressed
or suppressed in the SCC-9 and SCC-15 cell lines by infection with
lentiviruses expressing the Beclin1 (LV-Beclin1), Beclin1 shRNA1
(LV-shBec-1) and Beclin1 shRNA2 (LV-shBec-2) (Fig. 1). After viral
infection, more than 95% of the cells were GFP-positive, indicating
a high efciency of Beclin1 or shRNA delivery. The Beclin1 expression in LV-Beclin1 group was signicantly higher than in SCC-9 and
SCC-15 cells without transfection (p < 0.05) (Fig. 1AC). On the
other hand, the expression of Beclin1 are efciently silenced in
LV-shBec-1 and LV-shBec-2 groups as compared to normal control
and non-silencing group (p < 0.05) conrmed by qRT-PCR and western blot (Fig. 1DF). Meanwhile, the expression of autophagyrelated proteins, ATG4, ATG5 and LC3II were signicantly
enhanced in LV-Beclin1 group while decreased in LV-shBec-1 and
LV-shBec-2 groups (Fig. 2).
Effects of Beclin1 on cell growth, migration and invasion in TSCC cells
Frequent down-regulation of Beclin1 mRNA and protein in
TSCC suggested a potential anti-oncogenic role of this gene. To

985

investigate the possible anti-proliferative effects of Beclin1

in vitro, a MTT assay was performed and a cell growth curve was
generated. As shown in Fig. 3, the proliferation of SCC-9 and SCC15 cells infected with LV-Beclin1 was signicantly suppressed
(Fig. 3A and B). In contrast, Beclin1 shRNA transfected cells showed
signicantly increased viability relative to control group (Fig. 3A
and B).
To further evaluate the function of Beclin1 on TSCC cell migration and invasion, transwell chambers coated with or without
matrigel were utilized. We found that overexpressed Beclin1 led
to a signicantly decreased migratory and invasive ability when
compared with control group (p < 0.05, Fig. 3CF). However, cell
migration and invasion activity were dramatically enhanced when
Beclin1 expression was inhibited (p < 0.05, Fig. 3CF).

VEGF, MMP-2 and MMP-9 were involved in Beclin1-mediated

inhibition of proliferation and invasion
To investigate the potential mechanisms of Beclin1-mediated
inhibition of proliferation and invasion in TSCC cells, we further
determined the VEGF, MMP-2 and MMP-9 expression levels in cell
culture medium which are the functional proteins of cell proliferation and invasion. As shown in Fig. 4, The western blot revealed
that promotion of Beclin1 expression could markedly decrease
the productions of VEGF, MMP-2 and MMP-9 (p < 0.05), but silencing of Beclin1 effectively elevated the expressions of VEGF, MMP-2
and MMP-9 (p < 0.05) in respect to control. Similar results were
observed in ELISA experiments (Fig. 4EJ). However, mRNA expression levels of MMP2 and MMP9 had no obviously changed while

Fig. 1. Lentivirus delivery of Beclin1 and Beclin1-specic shRNAs to SCC-9 and SCC-15 cells. (A) SCC-9 and SCC-15 cells were transfected with empty vector (LV-GFP), Beclin1
(LV-Beclin1), non-silencing shRNA (LV-shCon) and two different Beclin1-specic shRNA (LV-shBec-1 and LV-shBec-2). Forty-eight hours after transfection, the cells were
analyzed by western blotting for Beclin1. (B) Densitometry analysis of western blots from six independent experiments, respectively, showing that Beclin1 expression in LVBeclin1 group was signicantly higher and Becin1 shRNA specically knockdowned Beclin1 expression in tongue squamous cell cancer cell lines. (C) Bar graph showing qRTPCR analysis of cells using primers specic for Beclin1 or GAPDH mRNA. The mRNA expression level of Beclin1 is up-regulated in the LV-Beclin1 group and is down-regulated
by two different Beclin1-specic shRNA as compared with controls in SCC-9 and SCC-15 cell lines. All data is presented as mean SD. All the experiments were repeated at
least 3 times.

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J. Weng et al. / Oral Oncology 50 (2014) 983990

Fig. 2. Effect of Beclin1 on expression of autophagy-related proteins in TSCC cells. (A and B) Protein expression levels of ATG4, ATG5 and LC3 were revealed by western blot
analysis in the TSCC cells transfected with LV-GFP, LV-Beclin1, non-silencing-shRNA and Beclin1 shRNAs. (CE) Levels of LC3, ATG4 and ATG5 protein were estimated as a
ratio of LC3-II, ATG4 and ATG5 correspondingly to GAPDH levels. ATG4, ATG5 and LC3II were signicantly enhanced in LV-Beclin1 group while decreased in LV-shBec-1 and
LV-shBec-2 groups compared with control group.

cells were treated with Lv-shBec1 and Lv-shBec2 or Lv-Beclin1, but

VEGF mRNA expression could be regulated by Beclin1 as shown in
Fig. 4KM. These data suggested that Beclin1 may regulate TSCC
cell invasion through the proteolytic degradation of extracellular
matrix (ECM).
Beclin1 inhibits tumor growth in xenograft tumors in vivo
The results showed that Beclin1 could suppress tumor progression in vitro, then, we further evaluated the effects of Beclin1 on
the tumorigenic phenotype and in particular its contribution to
tumor growth in vivo. SCC-9 cells infected with or without
LV-Beclin1 and LV-shBec-1 were injected into mice. The results
demonstrated that xenografts with lower expression levels of
Beclin1 grew faster than those transfected with empty vector control. Conversely, overexpression of Beclin1 in SCC-9 cell line inhibits tumor growth in vivo as compared to control group (p < 0.05)
(Fig. 5). On day 35, tumors were removed and immunohistochemistry was performed to conrm the expression levels of Beclin1,
ATG5, VEGF, MMP2 and MMP9 in xenograft. As shown in Fig. 5D,
Beclin1 and ATG5 staining intensity is strong in the LV-Beclin1
group while VEGF, MMP2 and MMP9 staining is weak, the converse
results were observed in the Beclin1-knockdown group.
Discussion
Autophagy is a genetically programmed process which cell
removes damaged proteins and organelles to limits their cumulative deleterious effects [8]. Emerging evidences suggested that
deregulation of autophagy contributes to the pathogenesis of different disease states, including neurodegenerative disorders, cardiomyopathy, skeletal myopathies, and infectious diseases [21].
Moreover, most of studies indicated that the suppression of
autophagy activity was frequently correlated with malignant pro-

gress raising the possibility that defects in cellular autophagy contribute to the development of cancer. Specically, malignant cells
often display lower basal autophagic activity than their normal
counterparts in many types of tumor, such as breast cancer [22],
ovarian cancer [23] and gastric cancer [24]. In rat liver carcinogenesis models, autophagic activity is decreased slightly at a pre-neoplastic stage and becomes more substantially diminished at a later
stage [25]. Similar results were also observed in our previous study
[20]. However, it remains unknown whether the decrease in autophagic activity observed in malignant cells is mechanistically
important or merely an epiphenomenon in malignant progress.
Fortunately, the highly conserved autophagy-related genes provide
the opportunity to use genetic approaches to investigate the role of
autophagy in the development of cancer [9,21,26]. Beclin1, the rst
downstream autophagy-execution gene, is a critical component in
the class III PI3 kinase complex (PI3KC3) that induces the formation of autophagosomes [12,26]. Our previous studies conrm that
Beclin1 expression is dramatically decreased in TSCC which is consistent with other studies. To further elucidate the cellular effects
of Beclin1 in TSCC cells, stable cell lines with overexpression and
silence of Beclin1 were established and conrmed by qPCR and
western blot. Then, cell proliferation was determined by MTT
assay. We showed that Beclin1 could signicantly suppress proliferation in TSCC cell lines and tumor growth in vivo. This is consistent with several previous reports that over-expression of Beclin1
inhibits cell proliferation in cervical [27] and lung [28] cancer cells.
However, Beclin1 also play a protective role for cancer cells when
treated with cisplatin (shown in Supplementary Fig. 1). These ndings suggested Beclin1 has distinct role in cancer cells dependent
on the stress which is in agreement with the physical role of
autophagy maintaining the cellular homeostasis.
Invasion and metastasis are the hallmarks of cancer and the
main causes of death in patients with advanced TSCC [29]. Active
cell migration is a critical step in the invasion and metastasis cascade of cancers. Since Beclin1 has been shown to regulate the

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J. Weng et al. / Oral Oncology 50 (2014) 983990

D
LV-Beclin 1

LV-GFP

SCC-9

LV-shCon

LV-shBec-1

LV-shBec-2

*
*

LV-Beclin 1

LV-GFP

SCC-15

LV-shCon

LV-shBec-1

LV-shBec-2

* *

F
LV-Beclin 1

LV-GFP

SCC-9

LV-shCon

LV-shBec-1

LV-shBec-2

*
*

LV-Beclin 1

LV-GFP

SCC-15

LV-shCon

LV-shBec-1

LV-shBec-2

* *

Fig. 3. Effects of Beclin1 on TSCC cells proliferation, invasion and migration. (A) Proliferation curves of SCC-9 and SCC-15 cell lines following transfection with recombinants
were determined using MTT assay. Values are normalized against the non-transfected control. (B and C) The invasion and migration ability of SCC-9 and SCC-15 cells was
evaluated by transwell chambers assay. The migrated cells were stained with DAPI and counted under bright-eld microscopy at 100 magnications. Representative photos
and quantitative data are shown. Data were mean SD values from three experiments, each performed in triplicate. Scale bars represent 50 lm.

activity of autophagy through its interactions with Vps34 and PI3

kinase in an mTor independent pathway [14,15], we suspected that
Beclin1 may suppress cell migration and invasion. In the present
study, the effects of Beclin1 on the abilities of TSCC cells to invade
and migrate were also investigated by transwell assay with or
without matrigel. The data demonstrated that Beclin1 could inhibit
migration and invasion of TSCC cells. These results are consistent
with the role of Beclin1 in other cancer types, in which increased
Beclin1 expression impairs cell migration of cervical [27] and
breast [30] cancers indicating that Beclin1 may regulate metastasis
associated genes. To further conrm the role of autophagy in invasion, cancer cells were also treated with ATG5 siRNA, the results
demonstrated that silence of ATG5 leads to enhanced migration
of cancer cells which is in agreement with Beclin1 silence (shown
in Supplementary Fig. 2). The degradation or destruction of extracellular matrix (ECM) and basement membrane is the crucial steps
in metastasis of cancers in which matrix metalloproteinases
(MMPs) play a major role. Moreover, MMPs are also essential to
each step of capillary formation and formation of vascular endothelial basement membrane [31]. Angiogenesis is the process of
forming new blood vessels and requires degradation of the vascular basement membrane and remodeling of the ECM in order to
allow cancer cells to migrate and invade into the surrounding tissue [32]. Several studies demonstrated MMPs is involved in the

angiogenesis through regulation of vascular endothelial growth

factor (VEGF) [31,33]. Moreover, studies demonstrated that stimulation of autophagy signals can inhibit angiogenesis indicating that
autophagy response could be used as a potential strategy to inhibit
growth of new blood vessels in cancers [34]. Therefore, we further
detected the expression of secreted MMPs and VEGF after Beclin1
overexpression and silence. Our results showed that MMP2,
MMP9 and VEGF were involved in Beclin1-mediated inhibition of
invasion in TSCC cells. To further investigate the effect of Beclin1
on expression of VEGF, MMP2 and MMP9, qPCR was also performed to detect the mRNA expression level of VEGF, MMP2 and
MMP9. However, Beclin1 seems no obviously effect on MMP2
and MMP9 mRNA expression levels. These data suggested that
Beclin1 may suppress the mRNA expression of VEGF at transcriptional or post-transcriptional level but not only a by-stander effect
at protein level. For the MMP2 and MMP9, qPCR results could not
exclude the possible by-stander effect. We think a further study
will be necessary to clarify the exact mechanism in future.
Taken together, these ndings indicated that Beclin1 served as
tumor suppressor in TSCC development. Interestingly, Tang et al.
showed that LC3, ATG9a, Beclin1 and ATG5 overexpression were
associated with a poor prognosis in patients with oral squamous
cell carcinoma (OSCC) in a series of studies. These ndings indicated increased expression of Beclin1 also has a crucial role in

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J. Weng et al. / Oral Oncology 50 (2014) 983990

Fig. 4. Effect of Beclin1 on VEGF, MMP-2 and MMP-9 expression. (A and B) The productions of VEGF, MMP-2 and MMP-9 in the culture media were analyzed using western
blot and immunosorbent assay. (C and D) Levels of VEGF, MMP-2 and MMP-9 protein were estimated as a ratio to levels of GAPDH. (EJ) The graphs show the enzyme amount
of individual experiments and group means of SCC-9 and SCC-15 cells. (KM) mRNA expression levels of VEGF, MMP2 and MMP9 were quantied by qPCR.

OSCC progression [3537]. More importantly, we also found there

were distinct expression pattern of Beclin1 in different TSCC tissue
samples and different area at same samples, which demonstrated
normal-like expression pattern, increased expression pattern and
decreased pattern (shown in Supplementary Fig. 3). Similar results
were observed in colorectal cancer reported by Koukourakis et al.
[38]. Therefore, further studies are necessary to clarify the role of
autophagy and Beclin1 in OSCC development by classied the

patients into three groups at least, including normal-like expression pattern, increased expression pattern and decreased expression pattern. We supposed that the loss of Beclin1 expression
denes poor prognosis by modulating cancer cell migration and
growth as shown in this manuscript, while excessive overexpression of Beclin1 also denes subgroups of tumors with aggressive
clinical behavior through protecting cancer cell against stress
induced by hypoxia, acidity and chemotherapy to facilitate

J. Weng et al. / Oral Oncology 50 (2014) 983990

989

B
A
LV-Beclin 1

LV-shBec-1

LV-GFP

SCC-9

LV-shCon

Fig. 5. Effect of Beclin1 on TSCC xenograft in nude mice. SCC-9 cells were transfected with GFP, Beclin1, non-silencing-shRNA and Beclin1 shRNA1 and nude mice were
inoculated subcutaneously with 1  107 cells at one site per mouse. The tumor mass (xenograft) volume was measured every week from week 1 to week 5. Data are expressed
as the (means SD) and represent ve independent experiments. (A) Photograph of xenografts dissected from nude mice after 5 weeks subcutaneous inoculation. (B) Tumor
growth curve showing a signicant growth tendency in Beclin1 shRNA1 group (p < 0.05). Conversely, tumor growth was delayed signicantly in Beclin1 transfected group as
compared to the control group (p < 0.05). (C) Tumor weights were measured at day 35 and similar results were observed. (D) Representative images of Beclin1, ATG5, VEGF,
MMP2 and MMP9 immunostaining in SCC-9 xenograft tumors. Scale bars represent 50 lm.

metastasis and therapy resistant. Collectively, we conclude that the

roles of Beclin1 and autophagy are dependent on cancer type,
stage, genetic background and microenvironment. Our ndings

provide the rst implication that Beclin1 is important for cell

proliferation, and regulates cell invasion through modulating the
production of VEGF, MMP-2 and MMP-9 in TSCC cells.

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J. Weng et al. / Oral Oncology 50 (2014) 983990

Conict of Interest Statement

None declared.
Acknowledgements
This work was supported in part by the National Natural
Science Foundation of China (81202136), Specialized Research
Fund for the Doctoral Program of Higher Education
(20120171120068), Guangdong Province Nature Science Foundation (10151008901000025), Guangdong Province Developing
Program (2009B050700024), Guangzhou Developing Program
(2012J5100008) and Fundamental Research Funds for the Central
Universities (11ykpy47).
Appendix A. Supplementary material
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.oraloncology.
2014.06.020.
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