CANCER BIOTHERAPY AND RADIOPHARMACEUTICALS

Volume 28, Number 10, 2013

Mary Ann Liebert, Inc.
DOI: 10.1089/cbr.2012.1386

Downregulated RhoBTB2 Expression Contributes

to Poor Outcome in Osteosarcoma Patients

Zhe Jin, Ya-Xin Han, and Xiao-Rui Han

Abstract

Purpose: Osteosarcoma is a malignant bone tumor. RhoBTB2 protein participated in various cellular activities
and influenced pathways responsible for cell cycle and apoptosis. To address its potential as a therapeutic target
for osteosarcoma, this study investigated the effect of RhoBTB2 expression on osteosarcoma tissue and cell.
Materials and Methods: Real-time PCR and immunohistochemistry analysis were performed to evaluate the
level of RhoBTB2 mRNA and protein in 121 osteosarcoma specimens. The relationship of RhoBTB2 expression
with clinicopathological parameters of osteosarcoma patients was analyzed using Chi-square test. In addition, a
plasmid expressing the RhoBTB2 gene was transfected into human osteosarcoma (HOS) cell using Lipofectamine
2000, and the effects of RhoBTB2 on HOS cell were investigated using 3-(4,5-dimethylthiazolyl)-2,5-diphenyl-
tetrazoliumbromide assay and flow cytometry.
Results: This study reports that RhoBTB2 protein is weakly expressed in osteosarcoma specimens, but highly in
normal parts of specimens. RhoBTB2 expression is significantly associated with primary location and local
recurrence of osteosarcoma. Overexpression of RhoBTB2 results in significant G1 phase arrest and apoptosis in
HOS cell.
Conclusion: Taken together, we identified the role RhoBTB2 in osteosarcoma tissue and cell. The results might
not only be of relevance for diagnosis and prognosis, but potentially also provide a novel target for osteosarcoma
therapies.

Key words: apoptosis, caspase, cell cycle, clinicopathological parameters, RhoBTB2

Introduction RhoBTB2 was a multifunctional protein. RhoBTB2 protein

participated in various cellular activities, such as transcrip-
8,9

O steosarcoma is a malignant bone tumor that typically

occurs in children, adolescents, and young adults.1,2 It is

tional regulation and protein degradation. Alteration of

RhoBTB2 levels influences pathways responsible for cell

the second most common primary malignancy of bone after
multiple myeloma, which accounts for 20% of primary bone
malignancies.3 Previous studies have clearly demonstrated
that multiple genetic alterations are responsible for the de-
velopment and progression of osteosarcoma like other human
cancers.4,5
Tumor suppressor gene RhoBTB2, initially named Deleted
in Breast Cancer 2 gene, was isolated from human chromo-
some 8p21.6 RhoBTB2 harbors an N-terminal RhoGTPase
domain, two zinc finger (BTB/POZ) domains, and a con-
served C-terminal domain with an uncharacterized func-
tion.7 A number of functional studies suggested that
10
cycle, apoptosis, cytoskeleton, and membrane-trafficking.
Ling et al.11 found that ectopic expression of RhoBTB2 in-
hibits migration and invasion in the context of human breast
cancer cell lines in vitro. Freeman et al.12 found that over-
expression of RhoBTB2 has a short-term positive influence on
cell cycle progression and a long-term inhibitory effect, and
its absence delays the onset of drug-induced apoptosis.
Wilkins and Carpenter13 provided evidence that RhoBTB2 is
itself a substrate for cullin3-based ubiquitin ligase complexes,
as treatment with proteasomal inhibitor MG132 or shRNA
ablation of cullin3 resulted in increased levels of RhoBTB2.
Hamaguchi et al.6 found that RhoBTB2 expression was

Department of Orthopaedics, The First Affiliated Hospital of China Medical University, Shenyang, China.

Address correspondence to: Zhe Jin; Department of Orthopaedics, The First Affiliated Hospital of China Medical University; 155 Nanjing
North Street, Heping District, Shenyang 110001, China
E-mail: jinzheFH@163.com

709
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RhoBTB2 INHIBITS OSTEOSARCOMA
silenced in 50% of breast and lung cancer cell lines. Loss of
RhoBTB2 has been identified in a wide range of carcinomas,
including bladder,14 stomach,15 lung,16 and larynx.17
To our knowledge, the roles of RhoBTB2 in osteosarcoma
remain unclear. Here to determine whether genetic alteration
of the RhoBTB2 gene on chromosome 8p21 could be in-
volved in osteosarcoma, we evaluated the level of RhoBTB2
in osteosarcoma samples by Real-time polymerase chain re-
action (PCR) and Immunohistochemical staining (IHC) and
compared its correlation with clinicopathological parameters
of osteosarcoma patients. Our results also showed that exo-
genous RhoBTB2 expression could inhibit the proliferation of
human osteosarcoma (HOS) cells.
Materials and Methods
Study population and ethics statement
One hundred and twenty-one samples of osteosarcoma
patients were recruited from the First Affiliated Hospital of
China Medical University in this study from July 1992 to July
2012. None of the patients underwent radiotherapy or che-
motherapy before operation. All patients approved the use of
tumor tissues for clinical research and gave written informed
consent for participation. The procedure was approved by
China Medical University Ethics Committee.
Real-time PCR
Total RNA was isolated using an RNeasy Mini Kit
(Biomed). First strand cDNA was reverse transcribed with
1 lg of total RNA, using TaKaRa Reverse Transcription Kit
(TaKaRa) and oligo (dT) 15 primers (TaKaRa). The resultant
cDNA was then used for quantitative PCR reactions. The
RhoBTB2 primers were: 5-ACCATGTGGTACCCAGAA
ATC-3 (sense) and 5-GTGGGACTTCCAGAACTGCA-3 (H + L) antibody at a concentration of 1:500. Cover slips were
(antisense). The house keeping genes, GAPDH and b-actin,
were used as internal controls for normalization of the re-
sults. The GAPDH primers were: 5-AGAAGGCTGGGGCT
CATTTG-3 (sense) and 5-CGATCCACACGGAGTACT
TGC-3 (antisense). The b-actin primers were: 5-CTCCCT
GGAGAAGAGCTACGA-3 (sense) and 5-GTGGGACTTCC
AGAACTGCA-3 (antisense). Amplification of RhoBTB2,
GADPH, and b-actin were performed with 1 cycle at 95 C for
10 minutes, and 40 cycles of 95 C for 15 seconds and 60 C for
60 seconds. Calculation of the relative expression of each
transcript was performed using the 2 - DDCt method.18
Immunohistochemical staining
Immunohistochemistry was used to detect the expression
of RhoBTB2 protein in osteosarcoma samples. The study
population included 121 patients as described above. IHC
was performed on 4-lm sections obtained from formalin-
fixed, paraffin-embedded blocks. Endogenous peroxidase
activity was blocked with 3% hydrogen peroxide for 30
minutes. Antigen retrieval was carried out in citrate buffer
(10 mM, pH = 6) for 30 minutes at 95 C. RhoBTB2 (Delta
Biolabs) at 1:20 was applied incubated at 4 C overnight.
Afterward, sections were incubated with a biotinylated
secondary antibody, and then exposed to a streptavidin
complex horseradish peroxidase (HRP). Positive reactions
were visualized with 3, 3-diaminobenzidine tetrahydro-
chloride (Sigma-Aldrich), followed by counterstaining with
JIN ET 710
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hematoxylin. Normal tissue was used as a control. Sections
treated without primary antibody were used as negative
controls. The positive percentage of counted cells was graded
semiquantitatively according to a four-tier scoring system:
negative ( - ), 0%*5%; weakly positive ( + ), 6%*25%; mod-
erately positive ( + + ), 26%*50% and strongly positive ( + + + ),
51%*100%.
Cell lines and cell culture
The HOS cell line, was obtained from American Tissue
Type Collection (ATCC) and maintained in Minimum Es-
sential Medium (Life Technologies) supplemented with 10%
(v/v) fetal bovine serum and antibiotics (100 units/mL of
penicillin and 100 mg/mL of streptomycin) at 37 C in a 5%
(v/v) CO2 incubator.
Transient transfection of eukaryotic expression plasmid
pEGFP-N1-RhoBTB2
pEGFP-N1-RhoBTB2 was a kind gift from Zhang Ying
(China Medical University). Cells were transfected with
pEGFP-N1-RhoBTB2 using Lipofectamine 2000 (Invitro-
gen) according to the manufacturers instructions. pEGFP-N1
was used as a mock control. Then cells were cultured in
normal condition for further analyze.
Immunofluorescent microscopy
Cells were fixed with 4% paraformaldehyde, permeabi-
lized with 0.5% Triton-X, and then blocked with 2% bovine
serum albumin (BSA) in phosphate-buffered saline (PBS).
The primary RhoBTB2 antibody (sc-87065; Santa Cruz Bio-
technology) was used at a 1:50 concentration, and the sec-
ondary antibody was Alexa Fluor 594 donkey anti-goat IgG
mounted using ProLong Gold antifade reagent (P36930; Life
Technologies). Cells were examined and photographed using
an Olympus CX71 fluorescence microscope (Olympus).
3-(4,5-Dimethylthiazolyl)-2,5-diphenyltetrazoliumbromide
assays
Cell viability was assayed using 3-(4,5-dimethylthiazolyl)-
2,5-diphenyltetrazoliumbromide ( MTT) assays (Sigma) at
different time points (0, 12, 24, 48, and 72 hours). Briefly,
Parental HOS cells or HOS cells that had been mock trans-
fected with pEGFP-N1 or transfected with pEGFP-N1-
RhoBTB2 were seeded, respectively in 96-well plates (1,500
cells/well). After the cells adhered to the surface of culture
vessel, 0.5 mg/mL MTT was added to each well. Four hours
later, cells were lysed with dimethyl sulfoxide and absorbance
rates were measured at 550560 nm using a microplate reader
(Bio-Rad). Each group was run in quadruplicate wells. Results
were representative of three individual experiments.
4-6-Diamidino-2-phenylindole staining assays
4-6-Diamidino-2-phenylindole (DAPI) staining was per-
formed to detect apoptotic cells. Briefly, cells in six-well plates
(1 105 cells/well) were fixed in 3% (v/v) formaldehyde
(Sigma) for 15 minutes and stained with DAPI (KeyGEN) for
30 minutes. Cells were then examined and photographed
using an Olympus CX71 fluorescence microscope (Olympus).
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Measurement of apoptotic cell death
Cells were harvested 48 hours after transfection, and im-
munostained with Annexin-V fluorescein isothiocyanate (FITC)
and propidium iodide (PI) according to the manufacturers
instructions (Apoptosis Detection Kit, KeyGEN). Data analysis
was performed using CellQuest software (BD Biosciences).
Detection of cell cycle changes by flow cytometry
The cells as described above were removed from plates by
trypsinization and pooled with cell culture supernatant
containing nonadherent cells. Cells were washed once with
PBS, fixed in cold 70% ethanol, and stored at - 20 C until
analyzed. For staining, 1 106 cells were washed in PBS and
stained in PBS with 50 lg/mL PI (KeyGEN), 200 lg/mL
boiled RNaseA, and 0.1% Triton X-100. Analyses were per-
formed on a BD FACScan flow cytometer (BD Biosciences).
Caspase assays
Caspase 3 and caspase 9 activity were measured using
Caspase colorimetric assay kits according to the manufacturers
instructions (KeyGEN). Briefly, cellular protein was extracted
with the lysis buffer supplied, and total protein concentrations
were determined using the Bradford protein assay. Caspase 3
or caspase 9 substrate was then added, and after 2 hours at
37 C in the dark, caspase 3 or caspase 9 activity was measured
using a microtiter plate reader at 405 nm. Data were recorded
from three independent experiments.
RNA isolation and reverse transcriptase-PCR
Total RNA was isolated from parental HOS cells or
HOS cells that had been mock transfected with pEGFP-N1
or transfected with pEGFP-N1-RhoBTB2 using the RNeasy
Mini Kit (Biomed). First strand cDNA was reverse tran-
scribed with 1 lg total RNA, using the TaKaRa Reverse
Transcription Kit (TaKaRa Dalian) and oligo(dT)-15 primers
(TaKaRa) according to the manufacturers instructions. The
primers of RhoBTB2 and GAPDH have been described in
Real-time PCR. Finally, products were resolved by 1% aga-
rose gel electrophoresis, and visualized by ethidium bromide
staining and a UV imaging system (UVP; LLC).
analyzed using Chi-square test. Survival rates were analyzed
using the Kaplan-Meier method. Coxs proportional hazard
model was used to identify significant factors correlated with
prognosis in multivariate analysis. p < 0.05 was considered
statistically significant.
Results
Detection of RhoBTB2 in osteosarcoma tissues
and normal controls
Real-time PCR was carried out to investigate the level of
RhoBTB2 mRNA in osteosarcoma specimens. As shown in
the results, the level of RhoBTB2 mRNA exhibited lower
in osteosarcoma tissue than that in paired normal tissue
( p < 0.05, Fig. 1). Based on the above study, we further de-
tected the expression of RhoBTB2 at protein level by IHC in
osteosarcoma tissue and normal tissue. The results showed
that the level of RhoBTB2 protein in osteosarcoma tissue
was also lower than normal tissue. The immunostaining
results also showed that RhoBTB2 expression was dis-
tributed to the cytoplasm of normal cells and cancer cells
(Fig. 2).
RhoBTB2 expression and its correlation with
clinicopathological parameters of osteosarcoma patients
RhoBTB2 protein was weakly expressed in osteosarcoma
specimens, but highly in normal parts of specimens (Fig. 2).
We grouped the 121 osteosarcoma patients into four groups
according to the level of RhoBTB2 expression. We then an-
alyzed the potential relationship between the expression of
RhoBTB2 and the clinicopathological characteristics of these
patients. The results are summarized in Table 1. RhoBTB2
expression was significantly associated with the following
clinicopathological parameters: primary location and local
recurrence ( p < 0.05). No correlation was found with other
clinicopathological characteristics, such as sex, histological
type, and surgery type ( p > 0.05). Follow-up information was
available on 121 patients with osteosarcoma for periods
ranging from 5 months to 8 years (median = 56 months).
Kaplan-Meier analysis showed that high RhoBTB2

Western blot assays

Protein extracts (60 lg each) were separated by sodium
dodecyl sulfate polyacrylamide gel electrophoresis (SDS/
PAGE) and transferred to polyvinylidene fluoride (PVDF)
membranes. Membranes were blocked with 5% (w/v) nonfat
milk in PBST, and then probed with the indicated primary
antibodies at 4 C overnight. Primary antibodies used in-
cluded: RhoBTB2 (sc-87065, Santa Cruz Biotechnology) and
b-actin (sc-130656, Santa Cruz Biotechnology). After the
membranes were washed, the membranes were incubated
with the appropriate secondary antibodies for 2 hours at
room temperature. Bound antibody was detected using an
enhanced chemiluminescence kit (Amersham Biosciences)
according to the manufacturers directions.

Statistical analysis
FIG. 1. The level of RhoBTB2 mRNA was lower in osteo-
The level of RhoBTB2 expression and its correlation with sarcoma cancer tissue than matched normal tissue ( p < 0.01).
clinicopathological parameters of osteosarcoma patients was GAPDH and b-actin were used as internal controls.
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FIG. 2. Immunohistochemical
staining for RhoBTB2 protein
in specimens. RhoBTB2 local-
ized to the cytoplasm. The
nuclei were counterstained
with hematoxylin. (A) Paired
normal tissue; (B) Osteo-
sarcoma cancer tissue; (C)
Negative control.

expression was a favorable prognostic marker for patients
( p < 0.05, Fig. 3). Coxs proportional hazard analysis indi-
cated that RhoBTB2 was an independent prognostic factor
for osteosarcoma ( p < 0.05, Table 2).
RhoBTB2-expressing HOS cell lines
We next investigated the consequence of exogenous
RhoBTB2 expression in HOS cells. As shown in Figure 4A
and B, the results of RT-PCR and western blot analysis
confirmed exogenous expression of RhoBTB2 in HOS cells
after transfection. EGFP + HOS cells also were measured by
immunofluorescence analysis. The results showed the local-
ization of RhoBTB2 and EGFP in RhoBTB2-expressing HOS
cells (Fig. 4C).
The inhibitory effect of RhoBTB2 on HOS cells in vitro
MTT assay was performed to detect the inhibitory effects of
RhoBTB2 on HOS cells. The growth curves obtained demon-
strated that RhoBTB2 inhibited the growth of HOS cells (Fig.
5A). In addition, the optimal time for RhoBTB2 was 48 hours.
Next, the effects of RhoBTB2 on cell cycle progression
were examined. HOS cells after RhoBTB2 transfection were
observed to arrest in the G1 phase of the cell cycle. As shown
in Figure 5B, the percentages of cells in the G1 phase were
32.27% 6.7% (Normal: untreatment), 36.77% 6.2% ( Mock:
pEGFP-N1 transfection), and 62.35% 8.6% (RhoBTB2:
pEGFP-N1-RhoBTB2 transfection), respectively. DAPI stain-
ing was also performed to detect morphological changes in
the nucleus after transfection. As shown in Figure 5C,

Table 1. Relationship Between RhoBTB2 Expression and Clinicopathological Parameters of Osteosarcoma

RhoBTB2 expression
Clinicopathological features N - + ++ +++ PR (%) v2 p

Sex 1.18 0.576

Female 48 31 6 6 5 35.4
Male 73 59 4 6 4 19.2
Primary location 12.03 0.042
Humerus 21 16 2 1 2 23.8
Radius 20 17 1 0 2 15.0
Femur 26 19 2 2 3 26.9
Tibia 21 15 2 4 0 28.6
Fibula 17 14 1 2 0 17.6
Others 16 9 2 3 2 43.8
Histological type 4.12 0.254
Osteoblastic 33 22 3 5 3 33.3
Chondroblastic 23 18 2 2 1 21.7
Fibroblastic 25 19 2 3 1 24.0
Dilated blood vessels 19 14 2 1 2 26.3
Others 21 17 1 1 2 19.0
Surgery type 3.57 0.194
Amputation 50 34 6 7 3 32.0
Limb sparing 71 56 4 5 6 21.1
Lung metastasis 2.92 0.412
- 64 51 3 6 4 20.3
+ 57 39 7 6 5 31.6
Local recurrence 8.30 0.038
- 48 29 6 7 6 39.6
+ 73 61 4 5 3 16.4
Tumor size 1.51 0.349
< 6 cm 57 43 6 5 3 24.6
6 cm 64 47 4 7 6 26.6

PR, positive rate; v2 value, Chi-square distribution.

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nuclear condensation was observed in the apoptotic cells

present, and nuclear fragmentation appeared within 48
hours. The percentage of apoptotic cells present was deter-
mined using flow cytometry to detect cells stained with
Annexin V-FITC and PI. As shown in Figure 5D, the per-
centage of apoptotic HOS cells induced by RhoBTB2 within
48 hours was 3.87% 0.27%, whereas 0.61% 0.08% of the
cells transfected with pEGFP-N1 and 0.75% 0.07% of cells
with no treatment were undergoing apoptosis. Caspase 3
and caspase 9 activity of transfected cells was also found to
be higher than in untransfected ones or mock transfected
ones (Fig. 5E, p < 0.05).

Discussion
RhoBTB2 is a candidate breast cancer suppressor gene and
is decreased in about 50% of breast cancer cells.19,20 More
FIG. 3. Effects of RhoBTB2 expression on survival rates
and more evidence showed that loss of RhoBTB2 was de-
among the osteosarcoma patients. Kaplan-Meier curves for
cumulative survival rate were obtained on patients with tected not only in breast cancer,6 but also in a wide range of
osteosarcoma, stratified according to the RhoBTB2 expres- carcinomas, such as bladder,14 stomach,15 lung,16 and lar-
sion. High: the osteosarcoma patients with high RhoBTB2 ynx.17 In the present study, we clearly demonstrated, for the
expression; Low: the osteosarcoma patients with low first time, that loss of RhoBTB2 existed in osteosarcoma tis-
RhoBTB2 expression. sues on both mRNA and protein level, whereas almost all of
normal tissues expressed RhoBTB2. To evaluate the signifi-
Table 2. Multivariate Analysis of Clinical cance of RhoBTB2 in osteosarcoma development and pro-
Variables for 121 Osteosarcoma Patients gression, we further analyzed the relationship between
downregulation of RhoBTB2 expression and clinicopatho-
Relative risk
logical parameters of patients. We identified that loss of
Clinicopathological parameters (95% CI) p value
RhoBTB2 is associated with primary location and local re-
Sex 1.08 (0.611.65) 0.352 currence of osteosarcoma. In previous study, Shi et al.21
Primary location 0.78 (0.381.22) 0.415 found that lower expression of RhoBTB2 was observed
Histological type 1.22 (0.741.97) 0.246 preferentially in poor tumor, node, metastasis (TNM) staging
Surgery type 0.75 (0.341.13) 0.443 and histological grading in bladder cancer samples. How-
Lung metastasis 1.52 (0.922.32) 0.178 ever, loss of RhoBTB2 expression in breast cancers had a
Local recurrence 0.62 (0.251.61) 0.347 stronger association with age of onset, tumor histological
Tumor size (6 cm) 1.55 (0.952.47) 0.154 types, and positive rate expression.19 We reckoned these
RhoBTB2 expression ( + * + ++) 6.56 (1.8413.25) 0.047
differences were caused by different tissues. In addition, we
CI, confidence interval. identified that RhoBTB2 expression was a favorable

FIG. 4. Confirmation of ex-

ogenous RhoBTB2 expression
in human osteosarcoma
(HOS) cells. (A) Reverse
transcriptase-polymerase
chain reaction and (B) west-
ern blot analysis of RhoBTB2
levels in cells. (C) Immuno-
fluorescence showed locali-
zation of RhoBTB2 and EGFP
in cells. Normal: Parental
HOS cells; MOCK: HOS cells
transfected with pEGFP-N1;
Transfection: HOS cells
transfected with pEGFP-N1-
RhoBTB2.
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FIG. 5. The antitumor ac-

tivity of upregulated
RhoBTB2 in HOS cells. (A) 3-
(4,5-dimethylthiazolyl)-2,5-
diphenyltetrazoliumbromide
assays were performed to
determine the percentage of
growth inhibition associated
with RhoBTB2 transfected
into HOS cells compared
with untransfected cells ( p <
0.05). (B) Cell cycle changes
determined by staining with
PI. Transfected
cells showed a G1 phase ar-
rest. (C) Morphological
changes observed in nuclei of
HOS cells stained with 4-6-
Diamidino-2-phenylindole.
Gray arrows indicate nuclear
condensation that has oc-
curred, while white arrows
indicate nuclear fragments
present. (D) Cells were
double-stained with Annex-
in-V/PI and flow cytometry
(FCM) analysis performed to
determine the percentage of
apoptotic cells. (E) Caspase
assays confirmed the in-
volvement of the intrinsic
apoptosis pathway. Sig-
nificant activation of caspase
3 and caspase 9 was detected
( p < 0.05). Normal: Parental
HOS cells; MOCK: HOS cells
transfected with pEGFP-N1;
RhoBTB2: HOS cells trans-
fected with pEGFP-N1-
RhoBTB2.
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RhoBTB2 INHIBITS OSTEOSARCOMA
prognostic marker for patients with osteosarcoma. Con-
sistent with previous study, Mao et al.19 found that the
survival rate of patients with RhoBTB2-positive breast
cancer was statistically higher compared with patients with
RhoBTB2-negative breast cancer. Multivariate Cox regres-
sion analysis also displayed that RhoBTB2 silencing was an
independent risk factor for breast cancer.19 These data in-
dicated that loss of RhoBTB2 expression may be a vital
factor in the development and prognosis of cancer.
Previous studies from tumor cell lines showed that ex-
pression of RhoBTB2 at the mRNA level was extinguished
in 58% of breast, 19 50% of lung,16 and 75% of bladder
tumor cell lines.14 The loss of RhoBTB2 expression was
proved in HOS cells in this work. Furthermore, to detect
the functions of RhoBTB2, we increased the level of
RhoBTB2 in HOS cells by transfection. In previous studies,
RhoBTB2 protein has an influence on transcripts residing
within pathways responsible for cell cycle control, protein
transport, apoptosis and cytoskeleton regulation. 9,22,23
RhoBTB2 levels increased during drug induced apoptosis,
and knockdown of RhoBTB2 by siRNA delayed the onset
of apoptosis.12 Mao et al.19 found that the expression of
wild type RhoBTB2 significantly promoted the apoptosis of
breast tumor cells by flow cytometry and HE staining. Ectopic
expression of RhoBTB2 inhibits cellular proliferation identified
in lung and breast cancer cells.6 In this work, we also con-
firmed upregulated RhoBTB2 induced apoptosis in HOS cells.
Freeman et al.12 found that overexpression of RhoBTB2 has a
short-term positive influence on cell cycle progression and a
long-term inhibitory effect. Consistent with previous studies,
we found RhoBTB2 could induce G1 arrest in HOS cells. Other
study asserted that RhoBTB2-mediated downregulation of
cyclin D1 was obligatory for cell cycle arrest.24 Further sup-
port for the roles in cell cycle regulation has been provided
with the identification of RhoBTB2 as a target of the E2F1
transcription factor.12 It has been demonstrated that E2F1 is
implicated in the transcription of genes necessary for DNA
replication and cell cycle progression and can also promote
apoptosis.25
Conclusion
Taken together, we identified the role RhoBTB2 in oste-
osarcoma tissue and cell. The present study shows that loss
of RhoBTB2 expression might play an important role in
osteosarcoma. RhoBTB2 expression is a good indicator for
the favorable prognosis of osteosarcoma patients. Over-
expression of RhoBTB2 could induce apoptosis and G1
phase arrest in osteosarcoma cell. The results might not
only be of relevance for diagnosis and prognosis, but po-
tentially also provide a novel target for osteosarcoma can-
cer therapies.
Acknowledgment
We thank Zhang Ying for providing pEGFP-N1-RhoBTB2
and his continued interest and support of this work.
Disclosure Statement
The authors declare that they have no proprietary, finan-
cial, professional, or other personal interest in any product,
JIN ET 715
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service, and/or company that could be construed as influ-
encing the position presented in this article.
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