Original contribution

Expression of Sox2 in human cervical carcinogenesis

Jing Ji PhD
a
, Peng-Sheng Zheng MD, PhD
a,b,

a
Department of Reproductive Medicine, the First Affiliated Hospital of Medical School, Xi'an Jiaotong University,
Xi'an 710061, The People's Republic of China
b
Section of Cancer Stem Cell Research, Key Laboratory of Environment and Genes Related to Diseases,
Ministry of Education of People's Republic of China, Xi'an 710061, The People's Republic of China
Received 27 August 2009; revised 12 November 2009; accepted 20 November 2009
Keywords:
Cervical cancer;
Sox2;
Cancer stem cells;
Carcinogenesis
Summary Sox2 is a key transcription factor for embryonic development and plays a critical role in
determining the fate of stem cells. Recently, Sox2 has been detected in several human tumors, indicating
a potential function in tumorigenesis. We initially reported remarkably increased nuclear Sox2 staining
in cervical carcinomas compared with normal cervix (P b .05). Furthermore, Sox2 staining was detected
in most tumorsphere cells isolated from fresh cervical cancer tissues but not among the differentiated
tumorsphere cells. When Sox2 was stably expressed in cervical cancer cells (SiHa and HeLa), Sox2-
overexpressing cells had increased proliferation, clonogenicity, and tumorigenicity in vitro and in vivo
than control cells. These results suggest that Sox2 may participate in carcinogenesis of cervical
carcinomas and may be a potential therapeutic target molecule for cervical cancers.
2010 Elsevier Inc. All rights reserved.
1. Introduction
Cervical cancer is the second major cause of cancer-
related mortality in women worldwide and accounts for 250
000 deaths each year [1]. Although several factors have been
linked with cervical carcinogenesis, the molecular mechan-
isms underlying cervical carcinogenesis are still poorly
understood. In recent years, emerging evidence has sug-
gested that tumorigenesis is dependent on a small subset of
cells within the tumor, termed cancer stem cells (CSCs).
CSCs have been identified and isolated from hematopoitic
malignancy and solid tumors, including glioblastoma, breast
cancer, and colon cancer [2-5]. Moreover, the relationship
between anomalous expression of critical stem cell tran-
scription factors such as Oct4, Sox2, and Nanog and
carcinogenesis has been studied in various cancers [6-8].
Sox2, located in chromosome 3q26.3, is a member of the
SOX (SRY-related high mobility group box) family, which
all contain an high mobility group (HMG) domain very
similar to that in the sex-determining gene SRY [9]. So far,
more than 20 members of the SOX gene family have been
identified that play an essential role in stem cell biology,
regulation of organ development, and cell type specification
[10-12]. For example, Sox2 is initially expressed in all
blastomeres of the 4-cell embryo and later becomes restricted
to the inner cell mass and epiblast and then to the germ cells.
In mouse embryonic stem (mES) cells, reduction of Sox2
expression is associated with loss of the pluripotent state and
a propensity for differentiation [13].
Several recent studies have demonstrated that the
transcription factor Sox2 is related to several human
malignant tumors. Sox2 is overexpressed in 41% of small
cell lung cancers and 43%of basal cell-like breast carcinomas

This research was supported by the National Natural Science

Foundation of China (30571951/C1702; 30725043/C1702).

Corresponding author. Department of Reproductive Medicine, the

First Affiliated Hospital of Medical School, Xi'an Jiaotong University,
Xi'an 710061, The People's Republic of China.
E-mail address: zpsheng@mail.xjtu.edu.cn (P. -S. Zheng).
www.elsevier.com/locate/humpath
0046-8177/$ see front matter 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.humpath.2009.11.021
Human Pathology (2010) 41, 14381447
[14,15]. Sox2 was also found to be involved in later events of
carcinogenesis, such as invasion and metastasis, rather than
the early progression of pancreatic intraepithelial neoplasias
(PanINs) [16]. However, reduced expression of Sox2 has also
been associated with a few carcinomas such as gastric cancer
and choriocarcinoma [17,18].
To clarify the function of Sox2 in cervical carcinogenesis,
we examined the expression of Sox2 in normal and
pathologic cervical tissues, as well as in cervical cancer
cell lines. We also compared the expression of Sox2 in
cervical cancer tumorspheres and its differentiated cells. We
further studied stably expressed Sox2 in SiHa and HeLa cells
and examined the function of Sox2 in cell proliferation,
clonogenicity, and tumorigenicity.
2. Materials and methods
2.1. Cell lines and cell culture
All cell lines used in this study were obtained from
American type culture collection and cultured in Dulbecco's
modified Eagle medium (DMEM) (Sigma, St. Louis, MO,
USA) supplemented with 10% fetal bovine serum (FBS),
penicillin, and streptomycin.
2.2. Tumor samples and primary cervical tumor-
spherse cultures
This study was approved by the local ethics committee.
Tissue samples were obtained from the Department of
Gynecology, First Affiliated Hospital of Xi'an Jiaotong
University (China). Samples were collected immediately
after surgical resection, then washed, minced, and digested
by collagenase IV (Sigma) at 37C, as previously described
[19]. After 16 hours of digestion, cells were grown in serum-
free stem cell medium containing DMEM/F12 supplemented
with basic fibroblast growth factor (bFGF) (Upstate),
penicillin, and streptomycin (Sigma).
2.3. Tumorsphere differentiation assay
To induce differentiation, the tumorspheres were seeded
on poly-L-lysinecoated coverslips and cultured under
differentiation conditions in DMEM with 10% FBS. Tumor-
sphere cells adhered on the surface of coverslips and were
cultured for 10 days for complete differentiation, with the
medium exchanged every 3 days.
2.4. Cervical cancer tissue microarray
The cervical cancer tissue microarray was commercially
purchased from the Chaoying Company (Xi'an, China). This
tissue microarray contains 40 cervical squamous cell
carcinomas, 30 cervical carcinomas in situ (CIN III), and
32 normal uterine cervices. Hematoxylin and eosin-stained
sections were used to demonstrate pathologic and cytologic
diagnosis. Clinical stage and histologic classification were
based on the International Federation of Gynecology and
Obstetrics classification system.
2.5. Immunostaining
Immunocytochemistry was performed to examine the
expression of Sox2 in cervical cancer cell lines, primary
cervical tumorspheres, and differentiated cells. Cells were
plated onto coverslips and fixed with 4% paraformaldehyde
for 30 minutes at room temperature, then treated with 0.3%
Triton-X100, 10% normal rabbit serum, and incubated with
Sox2 antibody (Y-17, Goat polyclonal, 1:100; Santa Cruz sc-
17320, Santa Cruz Biotechnology, Santa Cruz, CA) or
phosphate buffered saline (PBS) as negative control.
Paraffin-embedded cervical cancer tissue microarraysections
were deparaffinized in xylene and rehydrated through graded
ethanol. After antigen retrieval and blocking of nonspecific
reactions, sections were incubated sequentially with primary
antibody, biotinylated antigoat immunoglobulin G, and strepta-
vidin-peroxidase conjugate and 3,3-diaminobenzidine sub-
strate. Sections were counterstained with hematoxylin.
Immunohistochemical results of Sox2 expressed in cervical
tissues were evaluated by 3 investigators independently.
According to previous studies [20,21], immunoreactivity of
Sox2 was semiquantitatively graded as 0, less than 10% cells
reactive; 1+, 10% to 25% cells reactive; 2+, 26% to 50% cells
reactive; 3+, 51% to 75% cells reactive; and 4+, more than
75% cells reactive. Samples with a score of 0 were considered
negative, and the rest of the cases were scored as positive.
2.6. Polymerase chain reaction analysis
Total RNA was extracted using TRIzol Reagent (Invitro-
gen, Carlsbad, CA, USA) and treated with DNaseI. RNA
(0.5-2 g) was reverse transcribed at 42C for 1 hour using
MMLV reverse transcriptase and oligo d(T) primer (MBI,
Hanover, MD, USA). Two microliters of complementary
DNA was used as a template for amplification by polymerase
chain reaction (PCR). Real-time quantitative PCR was
performed in triplicate for each primer set and in each cell
sample using an iQ5 multicolor Realtime PCR Detection
System (Bio-RAD, Hercules, CA, USA).
2.7. Sox2 overexpression vector construction
and transfection
Human Sox2 open reading frame was amplified by
reverse transcription PCR using high fidelity polymerase Pfx
(Invitrogen, Carlsbad, CA, USA) from messenger RNA
(mRNA) isolated from U87 cells. The primers used are as
follows: Sox2F 5-GTT CTC GAG AGC ATG TAC AAC
1439 Expression of Sox2 in human cervical carcinogenesis
1440 J. Ji, P. -S. Zheng
ATG ATG GAG-3; Sox2R 5-GTT GAA TTC AGG CCC
TCA CAT GTG TGA GAG-3. For Sox2 expression vectors,
the Sox2 complementary DNAs were inserted into the Xho I/
EcoR I site of pIRSE2-EGFP to create pIRSE2-Sox2. In
addition, the pIRES2-EGFP vector without any insert was
used as a negative control vector.
Sox2 gene or control vector was transfected in cervical
cancer cells (SiHa and HeLa) using Lipofectamine 2000
(Invitrogen) according to the manufacturer's instructions.
After 24-hour transfection, cells were passaged at 1:10 into
fresh DMEM with 10% FBS in the presence of 1500 g/mL
of G418 for 3 weeks. Individual drug-resistant clones were
selected, pooled, expanded, and identified by PCR and
Western blot.
2.8. Western blot
Protein samples (20 g) underwent electrophoresis on
10% sodium dodecyl sulfate polyacrylamide gel electropho-
resis (SDS-PAGE) and transferred onto nitrocellulose
membranes, blocked in 5% milk in Tris buffer saline-
Tween-20 and then probed with antibodies to Sox2 or -
Actin (Santa Cruz Biotechnology) overnight at 4C. After
reaction with horseradish peroxidase-labeled secondary
antibodies, the proteins were detected by using SuperSignal
West Pico Trial Kit (Pierce, Rockford, IL, USA).
2.9. Cell viability assessment
Cell viability was assessed using 3-(4,5-dimethylthiazol-
2-yl)-2,5-diphenyl tetrazolium bromide (MTT; Sigma) dye
according to the standard protocol. The number of viable
cells was determined by measuring absorbance at 490 nm
(Bio-Rad). In addition, cell proliferation was evaluated on
days 1, 3, 5, and 7 after seeding by counting cells with a
Coulter counter.
2.10. Flow cytometry
Cells (1 10
6
) were harvested and washed with PBS,
followed by fixation with 70% ethanol overnight at 4C.
After washing with PBS twice, the cells were resuspended in
PBS with 50 g/mL propidium iodide and 10 g/mL RNase
A for 30 minutes at room temperature in the dark. Cells were
analyzed for DNA content by flow cytometry, and the cell
cycle phases were analyzed using ModFit LT software
(Verity Software House Inc., Topsham, ME, USA).
2.11. Cloning assay
One hundred cells plated on 100-mm cell culture dishes
(Becton Dickinson) were cultured for 3 weeks. The cloning
cells were fixed with methanol for 15 minutes at room
temperature and then stained by Giemsa solution. The clones
containing 50 or more cells were regarded as true clones.
2.12. Colony formation in softer agar
Cells (1 10
3
) were suspended in 1 mL of DMEM
containing 10% FBS and 0.33% agar (Sigma) and plated
onto the same medium containing 0.5% agar (3 mL/60 mm
Petri dish; Becton-Dickinson, New York, NY, USA).
Colonies with diameters greater than 0.2 mm were counted
at day 28. Statistical analysis was done using Student t test.
2.13. Tumorsphere formation assay
100 Sox2-transfected or vector-transfected cells were
seeded in 6-well plates in 1 mL of serum-free stem cell
medium previously described. Fresh medium (0.5 mL) were
added to each well every 3 days. The tumorspheres that
contained more than 50 cells in each well were analyzed on
day 12 for tumorsphere-forming ability.
2.14. Tumor xenograft experiment
The Sox2-transfected or vector-transfected cells (10
6
or
10
5
) were injected into the subcutaneous tissue in the dorsum
of 4 to 6-week-old female BALB/c-nude mice. Three
animals per group were used in each experiment. Tumors
were measured weekly using a Vernier caliper, and volume
was calculated according to the formula: /6 length
width
2
.
2.15. Statistical analysis
All experiments were repeated at least 3 separate times.
Data from all experiments were pooled, and the results were
expressed as mean SD. Differences in mean values were
analyzed by analysis of variance with SPSS13.0 statistical
Table 1 Sox2 immunopositivity in different pathologic
grades of cervical SCC
Tumor n Sox2 P
0 1+ 2+ 3+ 4+
Grade I 11 5 0 2 1 3 b.05
GradeII-III 29 4 2 5 6 12
Fig. 1 Sox2 is overexpressed in cervical cancer. (A) Western blot analysis of Sox2 expression in cervical cancer cell lines, positive control
mouse embryonal carcinoma cell lines F9, P19, U87, and tumorsphere (TS), differentiated tumorshphere cells (DTS); -Actin was the loading
control. (B) Immunohistochemical staining of Sox2 protein in paraffin-embedded sections of normal cervix, CIN III, and cervical carcinoma
tissues. (C) Bar chart showing the positive cases (percentage) of Sox2 expression in normal cervix, CIN III, and cervical carcinomas.
1441 Expression of Sox2 in human cervical carcinogenesis
1442 J. Ji, P. -S. Zheng
software (SPSS Inc, Chicago, IL). P b .05 was considered to
be statistically significant.
3. Results
3.1. Expression of Sox2 in cervical cancer cell lines
and tumors
To determine whether the stem cell marker Sox2 was
expressed in cervical cancers, we performed Western blot
(Fig. 1A) in 4 human cervical cancer cell lines, and 2 mice
embryonic carcinoma cell lines (P19 and F9) were used as
positive controls. Compared with P19 and F9, C33A and
SiHa showed clear expression. There was no expression of
Sox2 in HeLa and CaSKi cells, which was confirmed by
immunocytochemistry (Supplementary Figs. 1A and 2B).
To further assess the potential effect of Sox2 in cervical
cancers, Sox2 expression was examined in 32 normal
cervices, 30 CIN III, and 40 cervical carcinoma tissues by
immunohistochemistry (Fig. 1B). Sox2 staining only
presented in 8 (25.0%) of 32 normal cervices, and most
positive cells were basal cells in cervical epithelium. A
significantly higher frequency of Sox2 staining was observed
in the CIN III (25/30, 83.3%) and cervical cancers (31/40,
77.5%) (Fig. 1C). Furthermore, squamous carcinoma II and
III showed a relatively higher percentage of Sox2 when
compared with squamous carcinoma I (Table 1). No
correlation was found between Sox2 expression and other
clinical features, including age, International Federation of
Gynecology and Obstetrics stage, and pelvic lymph node
invasion (data not shown).
3.2. Expression of Sox2 in tumorspheres derived
from primary cervical cancer tissues
For tumorsphere culture, cells dissociated from fresh
tissues were plated in the serum-free medium as described in
Materials and Methods. The tumorsphere cells became
differentiated cells after being cultured in DMEM containing
10% FBS for 10 days, which showed the characteristics of
adherent growth. Some stem cell-related genes such as
Nanog and UTF1 were expressed in tumorspheres but not in
differentiated cells (Supplementary Fig. 2A). We detected
Sox2 protein in fresh tissue-derived cervical cancer tumor-
sphere cells and its differentiated cells. Sox2 staining was
found in the nuclei of cervical cancer tumorsphere cells but
not in differentiated cells (Fig. 2A). This phenomenon was
confirmed by Western blot (Fig. 1A).
3.3. Exogenous Sox2 promotes proliferation and
clonogenicity in cervical cancer cells
To investigate the effects of Sox2 protein on cervical
carcinoma cells, exogenous Sox2 was stably transfected in
cervical cancer cell lines (SiHa and HeLa). Abundant Sox2
was detected in pIRES2-Sox2 transfected cells by Western
blotting but not in the control vector transfected cells
(Supplementary Figs. 2C and 3A). Both cell growth curves
and MTT showed that Sox2-overexpressing cells grew much
faster than control cells (Fig. 2B, C and Supplementary Fig.
3B, 3C), suggesting that exogenous Sox2 could enhance cell
proliferation. As shown in Fig. 2D (and Supplementary Fig.
3D), exogenous Sox2 also led to increased clone numbers
and size in cloning formation assay (P b .001). These
observations indicated that Sox2 contributed to the prolifer-
ation and cloning ability of cervical cancer cells in vitro.
3.4. Exogenous Sox2 promotes tumorigenicity in
vitro and in vivo
Tumor stem cells resemble stem cells in their ability to
grow as spheres when cultured in conditions where they
could not attach to a solid substratum [3,22]. We investigated
the ability of SiHa-Sox2 cells to form tumorspheres in
serum-free stem cell medium. Notably, SiHa-Sox2 cells
formed tumorspheres after 10 days of culture (Fig. 3A).
Furthermore, as shown in Fig. 3B, SiHa-Sox2 cells formed
more and much bigger tumorspheres than SiHa-EGFP cells
did. About 60% of SiHa-Sox2 cells formed tumorspheres,
but only about 15% of SiHa-EGFP cells did, even if the latter
were cultured up to 20 days. Similar results were observed
between HeLa-Sox2 and HeLa-EGFP cells (Supplementary
Fig.4A and 4B). Therefore, exogenous Sox2 expression may
add to the stemness of cervical cancer cells. Similarly,
Sox2-overexpressing cells formed more foci in soft agar than
control cells (P b .001) (Fig. 3Cand Supplementary Fig. 4C).
These data suggest that exogenous expression of Sox2 in
SiHa and HeLa cells may enhance the tumorigenicity
in vitro.
To further confirm the tumorigenicity of Sox2-over-
expressing cells in vivo, a tumor formation assay was done
with BALB/c nude mice. As shown in Fig. 3D, both SiHa-
Sox2 (left side, red arrow) and SiHa-EGFP cells (right side,
Fig. 2 Effects of exogenous Sox2 on cell proliferation and clone formation. (A) Sox2 immunoreactivity was predominantly detected in the
nuclei of primary cervical tumorspheres compared with their differentiation products (A1, 400). Bar chart showed the positive cells
(percentage) of Sox2 expression in them (A2). (B) MTT assay of Sox2 and control transfected cells (SiHa-Sox2 and SiHa-EGFP). (C) In vitro
cell proliferation curve of Sox2 and control transfected cells. (D) Left, clone formation by Sox2 and vector transfected cells; right,
quantification (box plot) of clone numbers showing high/low values, 25/75 percentiles, and medians. *P b .001.
1443 Expression of Sox2 in human cervical carcinogenesis
1444 J. Ji, P. -S. Zheng
yellow arrow) formed palpable tumors after 7 weeks in 10
6
cell groups. In addition, after 10 weeks, SiHa-Sox2 cells
derived larger tumors with a mean tumor size of 85 13
mm
3
, compared with smaller tumors (26 5 mm
3
) derived
from SiHa-EGFP cells. More obvious results could be
obtained from HeLa-Sox2 cells (Supplementary Fig. 4D).
Fig. 4 Exogenous Sox2 induces cell proliferation and cell cycle accelerating in SiHa cell. (A) Immunochemical staining for Sox2 and Ki-67
in SiHa tumor xenografts (1000). (B) Cell cycle analysis of SiHa-Sox2 and control cells; percentages of cell in each phase of the cell cycle are
indicated. (C) Real-time PCR analysis of the mRNA levels of various cell cycle regulatory genes upon SiHa-Sox2 cells and control cells.
*P b .05.
Fig. 3 Tumor-forming ability of SiHa-Sox2 and SiHa-EGFP cells in vitro and in vivo. (A) Tumorsphere formation by SiHa-Sox2 cells. (B)
Tumorsphere sizes generated by SiHa-Sox2 and control cells; figures indicate magnifications. (C) Efficiency of soft agar focus formation (box
plot) by SiHa-Sox2 and control cells. (D) Tumor formation (left) and tumor growth curves (right) of SiHa-Sox2 (red arrow) and control cells
(yellow arrow) in female BALB/c nude mice. Each bar represents mean tumor volume SD. *P b .001.
1445 Expression of Sox2 in human cervical carcinogenesis
These data indicated that exogenous Sox2 expression
significantly enhances tumor formation in vivo.
3.5. Exogenous Sox2 induces cell cycle accelerating
in cervical cancer cells
To investigate the mechanisms by which Sox2 promotes
tumorigenecity, we examined Sox2 expression in xenograft
tumors (Fig. 4A and Supplementary Fig. 5A). Sox2 staining
is obviously higher in tumors with Sox2-overexpressing cells
than that in tumors with control cells. We also detected Ki-67
in the tumors formed in nude mice. The tumors formed by
Sox2-overexpressing cells showed more Ki-67 staining than
that by control cells. Second, the SiHa-Sox2 cells showed a
higher population of cells in the S phases (42.7%) than in the
control cells (25.9%) (Fig. 4B). A similar phenomenon was
seen in HeLa-Sox2 cells. The results of real-time PCR
showed the mRNA levels of CyclinE2, MCM10, and Wee1
were increased in the Sox2-overexpressing cells, and the
level of CyclinA1 was almost the same (Fig. 4C and
Supplementary Fig. 5C). These results suggested that Sox2
may promote cell proliferation and tumorigencity by
facilitating the G1 to S transition.
4. Discussion
Sox2 is a member of the SOX gene family that encodes
transcription factors with a single high mobility group
(HMG) DNA-binding domain [9]. The SOX family of
transcription factors expresses in various phases of embry-
onic development, which affects cell fate and differentiation
[11]. In the mouse, Sox2 is primarily expressed during
embryogenesis, where it has been shown to be developmen-
tally regulated. Sox2-null mutant embryos die around
implantation [23], indicating that Sox2 plays an essential
role in early embryo precursor cells. In vitro, Sox2 is
expressed in undifferentiated embryonic stem cells and
embryonal carcinoma cells and is down-regulated when
these cells are induced to differentiate by retinoic acid or the
removal of leukemia inhibitory factor in culture medium[24].
These remarkable expression patterns of Sox2 suggest that
Sox2 plays an important role in maintaining the pluripotency
of cells in self-renewal and differentiation potential.
Recently, Sox2 has been studied in several types of human
solid tumors. In human breast cancer and pancreatic
carcinoma [16,25], Sox2 is overexpressed, and the protein
level is correlated with tumor pathologic grade. However, in
gastric cancers and choriocarcinomas [17,18], Sox2 was
down-regulated by aberrant DNA methylation. In the present
study, for the first time, we illustrated the expression of Sox2
in normal and pathologic cervical tissues, as well as cervical
cancer cell lines. First, we observed clear nuclei expression of
Sox2 in SiHa and C33A but not in HeLa and CaSki. CaSki is
an epidermoid carcinoma, one kind of squamous cell
carcinoma, and SiHa is a squamous cell carcinoma, so we
think the expression of Sox2 might be related to the cancer
type. SiHa, HeLa, and CaSki are positive cell lines for human
papillomavirus (HPV), but C33A is negative for HPV. In our
results, it seems HPVinfection does not showany association
with the expression of Sox2. Second, Sox2 staining
was found in 8 (25.0%) of 32 normal cervixes, 25 (83.3%)
of 30 CIN III, and 31 (77.5%) of 40 cervical squamous
carcinomas, which indicates that Sox2 expression may be
associated with cervical carcinogenesis. Sox2 staining in
normal cervical epithelium was found only in the basal cells,
where epithelial reserve cells were located. Whether these
Sox2 positive cells are stem cells in normal cervical epithelia,
which was thought by Peters [26] and Smedts [27] more than
20 years ago, or these positive cells are initiating cells for
precancerous lesions requires further study.
Prasad [28] reported that Sox2 is associated with early
events of carcinogenesis, such as the progression from
normal epithelium to PanIN-2. Squamous cell carcinomas
are divided into 3 grades. Grade I is well differentiated
(squamous cell carcinoma I), grade II is moderately
differentiated (squamous cell carcinoma II), and grade III
is poorly differentiated (squamous cell carcinoma III). Our
studies indicate that the Sox2 gene might contribute to
tumorigenesis of cervical cancer with the following evi-
dence: (1) about 80% of CIN III or cervical squamous
carcinoma expressed Sox2 protein, but it is expressed in only
25% of normal cervix; (2) squamous carcinoma II and III
showed a relatively higher intensity of Sox2 staining
compared with that of squamous carcinoma I (P b .05); (3)
Sox2 was clearly and strongly expressed in the primary
tumorspheres derived from fresh cervical cancer tissues but
never or seldom detected in the differentiated cells. These
results are consistent with previous studies in which Sox2
staining was found in glioblastoma tumorspheres [7].
To further explore the effects of Sox2 in cervical
carcinogenesis, exogenous Sox2 was stably expressed in
SiHa and HeLa cells. In the present study, we found that both
Sox2-overexpressing and control cells could form tumor-
spheres in serum-free stem cell medium. However, both the
size and number of tumorspheres formed by SiHa-Sox2 cells
were much greater than the tumorspheres formed by control
cells. These results indicate that exogenous Sox2 expression
partly assigned the characteristics (such as tumorsphere
formation) of tumor initiating cells to Sox2-negative cells.
We also found that exogenous Sox2 expression could promote
both cell proliferation and growth identified by cell growth
curves, MTT, and cloning formation assay. At the cellular
level, it appeared that exogenous Sox2 promoted cell cycle
progression by facilitating the G
1
to S transition. Sox2
expression also enhanced cells' anchorage-independent
growth in soft agar and tumor formation in nude mice. Similar
results were obtained from the HeLa-Sox2 and HeLa-EGFP
cells. These data suggest that exogenous Sox2 expression
might enhance cervical tumorigenesis both in vitro and in vivo.
Chen [26] reported similar results in breast cancer.
1446 J. Ji, P. -S. Zheng
In summary, we have first reported that expression of
Sox2, a stem cell specific transcriptional factor, was
significantly higher in cervical cancers compared with
human normal cervix. In cervical squamous carcinoma,
Sox2 expression levels correlated with tumor pathologic
grade. Overexpression of Sox2 increased proliferation,
clonogenicity, and tumorigenicity in cervical cancer cells.
These results demonstrate that Sox2 may play an important
role in tumorigenesis and suggest a possible therapeutic
target molecule, in cervical cancers.
Appendix A. Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.humpath.2009.
11.021.
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1447 Expression of Sox2 in human cervical carcinogenesis