Upregulation of HYAL1 Expression in Breast Cancer
Promoted Tumor Cell Proliferation, Migration, Invasion
Jin-Xiang Tan1,2, Xiao-Yi Wang1,2., Xin-Liang Su1,2., Hong-Yuan Li1,2, Yuan Shi3, Liang Wang4,
1Department of Endocrine and Breast Surgery, the First Affiliated Hospital of Chongqing Medical University, Chongqing, China, 2Molecular Oncology and Epigenetics
Laboratory, the First Affiliated Hospital of Chongqing Medical University, Chongqing, China, 3Department of Anaesthesiology, Children’s Hospital of Chongqing Medical
University, Chongqing, China, 4Department of General Surgery, the First Hospital of Jiulongpo District, Chongqing, China
Hyaluronic acid (HA) is a component of the Extra-cellular matrix (ECM), it is closely correlated with tumor cell growth,
proliferation, metastasis and angiogenesis, etc. Hyaluronidase (HAase) is a HA-degrading endoglycosidase, levels of HAase
are elevated in many cancers. Hyaluronidase-1 (HYAL1) is the major tumor-derived HAase. We previously demonstrated that
HYAL1 were overexpression in human breast cancer. Breast cancer cells with higher HAase expression, exhibited
significantly higher invasion ability through matrigel than those cells with lower HAase expression, and knockdown of
HYAL1 expression in breast cancer cells resulted in decreased cell growth, adhesion, invasion and angiogenesis. Here, to
further elucidate the function of HYAL1 in breast cancer, we investigated the consequences of forcing HYAL1 expression in
breast cancer cells by transfection of expression plasmid. Compared with control, HYAL1 up-regulated cells showed
increased the HAase activity, and reduced the expression of HA in vitro. Meantime, upregulation of HYAL1 promoted the
cell growth, migration, invasion and angiogenesis in vitro. Moreover, in nude mice model, forcing HYAL1 expression
induced breast cancer cell xenograft tumor growth and angiogenesis. Interestingly, the HA expression was upregulated by
forcing HYAL1 expression in vivo. These findings suggested that HYAL1-HA system is correlated with the malignant
behavior of breast cancer.
Citation: Tan J-X, Wang X-Y, Su X-L, Li H-Y, Shi Y, et al. (2011) Upregulation of HYAL1 Expression in Breast Cancer Promoted Tumor Cell Proliferation, Migration,
Invasion and Angiogenesis. PLoS ONE 6(7): e22836. doi:10.1371/journal.pone.0022836
Editor: Roger Chammas, Faculdade de Medicina, Universidade de Sa ˜o Paulo, Brazil
Received February 11, 2011; Accepted June 29, 2011; Published July 28, 2011
Copyright: ? 2011 Tan et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by National Natural Science Foundation of China (Number: 30371393). The funders had no role in study design, data
collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org
. These authors contributed equally to this work.
Extra-cellular matrix (ECM) is closely correlated with tumor
progression. Hyaluronic acid (HA) is a component of the ECM, it
is an unsulfated anionic linear glycosaminoglycan polymer
comprised of a repeating glucuronic acid and N-acetylglucosamine
disaccharide motif . HA keeps tissues hydrated, maintains
osmotic balance and cartilage integrity [2–3]. HA also actively
regulates cell adhesion, migration, and proliferation by interacting
with specific cell surface receptors such as CD44 and RHAMM
. The concentration of HA is elevated in several inflammatory
diseases and various carcinomas, including bladder, prostate,
breast, lung, colon, and so forth [5–10]. Small fragments of HA,
generated by Hyaluronidase (HAase), stimulate angiogenesis
[11,12]. In tumor tissues, it may promote tumor growth and
metastasis probably by actively supporting tumor cell migration
and offering protection against immune surveillance .
HAases are a class of enzymes that predominantly degrade HA,
they are endoglycosidases, as they degrade the b-N-acetyl-D-
glucosaminidic linkages in the HA polymer . HAase has been
shown to alter the expression of CD44 isoforms, which may also
be involved in tumor progression . In addition, HAase is
associated with increased tumor cell cycling . The HAase
levels serve as an accurate marker for detecting prostate and
bladder tumors [9–10]. In humans, six HAase genes have been
identified. Hyaluronidase-1 (HYAL1) was originally purified from
human plasma and urine [16–17], it is the major tumor-derived
HAase expressed in bladder and prostate cancer tissues, and it has
characterized expression at the mRNA and protein levels in tumor
cells [9,10]. HYAL1 is a ,55–60 kDa protein, and it is consisted
with 435 amino acids. An elevated level of HYAL1 has been found
in prostate, bladder, breast, head and neck cancers, etc [9–10,18–
20]. HYAL1 was the first HAase to be recognized as being
expressed by tumor cells and its expression correlates with their
invasive and metastatic potential . No HYAL1 expression is
observed in the tumor-associated stroma, although HYAL1
expression appears to correlate and perhaps induce HA produc-
tion in the tumor-associated stroma [21–22].
Among the six HAases, HYAL1 and Hyaluronidase-2 (HYAL2)
are widely distributed to degrade high molecular weight (MW) HA
. The HYAL2 cleaves high MW HA into ,20 kDa HA
fragments , which are transported intracellularly and further
PLoS ONE | www.plosone.org1July 2011 | Volume 6 | Issue 7 | e22836
digested into low MW HA fragments by HYAL1 . The small
angiogenic HA fragments stimulate endothelial cell proliferation,
adhesion and migration by activating the focal adhesion kinase
and mitogen-activated protein kinase pathways . HYAL1 has
been found as an independent predictor of biochemical recurrence
. HAase levels also increase in breast cancer cells when they
become metastatic .
We previously demonstrated that HYAL1 protein and activity
were overexpression in breast cancer tissues and cells [19,28], and
breast cancer cells with higher HAase expression, exhibited
significantly higher invasion ability through matrigel than those
cells with lower HAase expression . Knockdown of HYAL1
expression in breast cancer cells resulted in decreased cell growth,
adhesion, invasion and angiogenesis [19,29]. In this study, to
further elucidate the function of HYAL1 in breast cancer, we
demonstrated that forcing expression of HYAL1 in breast cancer
cells promoted tumor progression in vitro and in vivo. We
therefore provided functional evidence that HYAL1 is oncogenic
for breast cancer and functional antagonism of HYAL1
constitutes a potential therapeutic strategy for HYAL1 positive
Identification of the recombinant plasmid
The HYAL1 expressing plasmids were constructed using the
eukaryotic expression vector pcDNA3.1(+). The positions of the
restriction enzyme digestion sites were marked (Fig. 1A). After
sequence analysis, transfection of the recombinant plasmid
pcDNA3.1-HYAL1 or pcDNA3.1 empty vector into MCF7 cells,
respectively. After stably transfected cells were individually
selected, the HYAL1, HYAL2 and the housekeeping gene b-actin
mRNA levels were measured using RT-PCR. Compared with the
control group, the pcDNA3.1-HYAL1 transcripts were sharply
up-regulated (Fig. 1B). Accordingly, HYAL1 protein levels were
increased in pcDNA3.1-HYAL1 transfected cells too, as indicated
by western blotting (Fig. 1C) and immunofluorescence (Fig. 1D)
analyses. The pcDNA3.1 empty vector did not substantial affect
the endogenous HYAL1 expression, and there was no difference in
the HYAL2 expression among groups. These results demons-
trated that pcDNA3.1-HYAL1 was up-regulated specifically and
Upregulation of HYAL1 in breast cancer cells increased
colony formation and enhanced cell proliferation
To determine the functional consequences of forcing HYAL1
expression in human breast cancer cells, we generated the breast
cancer cell lines MCF7 and ZR-75-30 with upregulation of
HYAL1 by stable transfection with HYAL1 expression vector
pcDNA3.1 vector (designated MCF7-Vec and ZR-Vec), respec-
tively. Soft agar assays were then performed to determine
whether upregulation of HYAL1 could enhance colony forma-
tion in an anchorage-independent culture system. In soft agar
assays, the number of colonies of MCF7-HYAL1 and ZR-
HYAL1 were 42.264.6 and 34.964.8, however, the number of
colonies of MCF7-Vec and ZR-Vec were 31.165.4 and
26.263.6, respectively. There was a statistically significant
increase in the number of colonies of pcDNA3.1-HYAL1
compared to pcDNA3.1-Vec control cells (Fig. 2A; p,0.01). In
3D Matrigel cultures, the size (mm) of colonies of MCF7-HYAL1
(53.2616.0) and ZR-HYAL1 (48.8621.5) were bigger than
MCF7-Vec (38.3612.8) and ZR-Vec (39.1610.9), respectively
(Fig. 2B; p,0.05). Then, we examined the proliferation activity
of the transfected cells using MTT assay. As shown in Fig. 2C,
compared with the controls, the proliferation rates of MCF7-
HYAL1 and ZR-HYAL1 transfected cells were increased
significantly (p,0.01). These results indicated that HYAL1 is
correlated with cell proliferation.
ZR-HYAL1)or the empty
Upregulation of HYAL1 expression increased HAase
activity, and reduced HA expression
HAase ELISA-like assay was carried out as described
previously. As shown in Fig.3A, the amount HAase activity
(mU/mg) in MCF7-HYAL1 (14.7862.59) and ZR-HYAL1
(12.6962.78) were higher than MCF-Vec (5.3461.07) and ZR-
Vec (4.6061.09), respectively (p,0.01). However, compare with
MCF-Vec (0.5060.10) and ZR-Vec (0.4360.11), the HA levels
(0.2660.08) were decreased, respectively (Fig.3B; p,0.05). These
results demonstrated that pcDNA3.1-HYAL1 can enhance the
HAase activity, and the HYAL1 could degrade HA.
Upregulation of HYAL1 expression promoted breast
cancer cell cycling
To determine whether HYAL1 regulates cell cycle progression
induced by growth factor, Flow cytometry was performed to
analyze cell cycle phases. Compared with MCF7-Vec and ZR-
Vec, both in MCF7-HYAL1 (Fig. 4A) and ZR-HYAL1 (Fig. 4B)
transfectants, the number of cells in G0/G1 phase were
decreased, but those in S phase were increased (p,0.05,
Figure 1. Identification of the recombinant plasmid. Construc-
tion of the recombinant eukaryotic expression vector pcDNA3.1(+)
containing the HYAL1 gene used in the studies. The positions of the
restriction enzyme digestion sites were marked (A). Transfection of the
recombinant plasmid pcDNA3.1-HYAL1 or pcDNA3.1 empty vector into
MCF7 cells, respectively. RT-PCR (B), Western blot (C) and immunoflu-
orescence (D) analysis validated the successful transfection and the
expression of the HYAL1 mRNA and protein were up-regulated in
pcDNA3.1-HYAL1, the pcDNA3.1-HYAL1 did not substantially affect the
HYAL1 and Breast Cancer
PLoS ONE | www.plosone.org2 July 2011 | Volume 6 | Issue 7 | e22836
p,0.01, respectively). No alterations in G2/M phase were
observed (p.0.05). These results indicated that HYAL1 is
correlated with cell cycle in vitro.
Upregulation of HYAL1 expression enhanced the cell
migration, invasion and angiogenesis potential of breast
One of the most distinct features of breast cancer cell is the
invasive growth pattern and promotion in angiogenesis, which
prevents total surgical resection. Therefore, we evaluated the effect
of HYAL1 upregulation on cell migration, invasion and
To study the effect of HYAL1 upregulation on the cell
migration, wound-healing assays were carried out by allowing
the cells to move to the scar region for 24 h and 48 h. We
observed that forced expression of HYAL1 in both MCF7 and
ZR-75-30 cells did indeed stimulate wound closure compared with
their respective vector-transfected cells (Fig. 5A and B; p,0.05,
Invasion assays were performed using ECM gel-coated transwell
culture chambers. After 48 h of incubation, invading cells were
counted. We observed that forced expression of HYAL1 in both
MCF7 and ZR-75-30 cells did indeed stimulate cell invasion
compared their respective vector-transfected cells (Fig. 6A;
p,0.01). The potential of promoting angiogenesis was analysed
by in vitro angiogenesis assay. CRL-1730 cells formed branch-like
or net-like structures when co-cultured with MCF7 and ZR-75-30
cells, which were not seen in the absence of tumor cells. The
numbers of branch-like and net-like structures formed by CRL-
1730 co-cultured with MCF7 cells were acutely increased in the
MCF7-HYAL1. Similarly, in the ZR-75-30 and CRL-1730 co-
culture system, compared with ZR-Vec, the numbers of branch-
like and net-like structures in ZR-HYAL1 group was increased
obviously. (Fig. 6B; p,0.05). These results demonstrated that
HYAL1 is correlated with cell migration, invasion and angiogen-
esis potential of breast cancer cells.
Figure 2. Upregulation of HYAL1 in breast cancer cells increased colony formation and enhanced cell proliferation. MCF7-HYAL1 and
ZR-HYAL1 enhanced the number of colony formation in Soft agar (A) and size of colony in 3D Matrigel (B) (p,0.01, p,0.05, respectively), (original
magnification 6100). MTT assay showed that MCF7-HYAL1 and ZR-HYAL1 increased cell proliferation rates (C; p,0.01).
Figure 3. Summary of HAase activity and HA expression by
ELISA-like assay. In MCF7 and ZR-75-30 cells, the HAase activity was
increased significantly by pcDNA3.1-HYAL1 transfectants (A; p,0.01).
However, the HA expression was decreased (B; p,0.05).
HYAL1 and Breast Cancer
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Upregulation of HYAL1 expression promoted the
tumorigenesis of breast cancer cells in vivo
To evaluate the tumorigenetic capability of HYAL1 upregula-
tion tumor cells in vivo, 20 mice were injected into the mammary
fat-pad with 16107MCF7 cells, and the tumor growth was
monitored for a period of 9 weeks. These mice were randomly
divided into 2 groups (MCF7-Vec and MCF7-HYAL1) with 10
mice in each group. All of mice developed tumors at the end of the
experiment. However, the mice treated with MCF7-HYAL1 cells
showed a significant enhancement of tumor growth compared
with those treated with MCF7-Vec cells (Fig. 7A; p,0.01). 9 weeks
after inoculation, the average tumor weight in MCF7-HYAL1
(3.1860.64 g) was heavier than MCF7-Vec (1.9160.49 g) (Fig. 7B;
p,0.01). Western blot (Fig. 7C) and immunohistochemistry
(Fig. 7D) analysis confirmed that the HYAL1 protein expression
was significant enhanced in the MCF7-HYAL1 group. The
HYAL2 was not changed obviously. Ki67 is a molecular maker
that is related to proliferation, was located in the nucleus.
Compared with MCF7-Vec, the ki67 was highly expressed in
MCF7-HYAL1 group (Fig. 7D). CD31 is an endothelial marker,
Figure 4. Upregulation of HYAL1 effected on cell cycle. The MCF7-HYAL1 (A) and ZR-HYAL1 (B) transfectants had a higher portion in S phase
and a lower portion in G0/G1phase ((p,0.05, p,0.01, respectively)). No alterations in G2/M phase were observed (p.0.05).
Figure 5. Upregulation of HYAL1 in breast cancer cells promoted cells migration in vitro. The MCF7-HYAL1 (A) and ZR-HYAL1 (B)
transfectants promoted wound closure compared with their respective empty vector transfected cells (p,0.05, p,0.01, respectively), (original
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so the microvessel density (MVD) of the tumor tissue was assessed
by CD31 immunohistochemistry analysis. Compared with MCF7-
Vec, the expression of CD31 in MCF7-HYAL1 was increased
obviously (Fig. 7D). In addition, compared with MCF7-Vec
(7.2661.56) group, the HAase activity (mU/mg) in the MCF7-
HYAL1 (18.4263.23) group was enhanced significantly (Fig. 7E;
p,0.01). At same time, the HA expression level (mg/mg) in MCF7-
HYAL1 (15.6963.63) was higher than MCF7-Vec (11.7562.21)
(Fig. 7F; p,0.05). These data indicated that HYAL1 could
promote tumor proliferation and angiogenesis in vivo, and the HA
expression was increased in tumor tissue during enhancing tumor
malignant potentiality, although the HYAL1 could degrade HA.
In this study, the eukaryotic expression plasmid pcDNA3.1-
HYAL1 was constructed to force HYAL1 expression in breast
cancer cell lines MCF7 and ZR-75-30. Our results showed that
upregulation of HYAL1 resulted in cell growth increase in vitro
and in vivo (Fig. 2, 7A and 7B). It was also identified that HYAL1
expression in bladder cells regulated tumor gowth . These
results suggested that HYAL1 expression in tumor cells is required
for cell proliferation. Meanwhile, upregulation of HYAL1
expression enhanced the proportion of cells cycling in S phase
(Fig. 4), which is consistent with Lin et al.  and our previous
researches [19,29]. Based on the analysis of cell cycle regulators,
HYAL1 affects cell proliferation probably by regulating cell cycle.
Our finding that upregulation of HYAL1 in breast cancer cells
could enhance the HAase activity significantly (Fig. 3A), and the
HA expression was decreased obviously (Fig. 3A) in vitro, these
results identified that HYAL1 could degrade HA. Which was
according with previous researches [19,25]. Interestingly, upregu-
lation of HYAL1 expression enhanced the HAase activity (Fig. 7E),
at the same time, the HA expression was increased (Fig. 7F) in
vivo. Lokeshwar et al.  found that high level of HA was
expression in tumor-associated stroma of HYAL1-sense tumor
specimen, but very low HA expression was observed in the stromal
compartment of HYAL1-antisense tumor specimens. Which
indicated the HYAL1 could induce the stroma cells of tumor to
secrete HA, although it could cleave HA.
In addition to the effect of HYAL1 on tumor growth, its effects
on tumor cell migration and invasion are interesting. Our previous
researches showed that breast cancer cells with higher HAase
expression, exhibit significantly higher invation ability through
matrigel than those cells with lower HAase expression .
Knockdown of HYAL1 expression in breast cancer cells resulted
in decreased cell invasion . HYAL1 was also an independent
prognostic indicator for predicting biochemical recurrence in
prostate cancer and increased metastatic potential in a prostate
cancer model . In the current study, we demonstrated that
upregulation of HYAL1 expression in MCF7 and ZR-75-30 cells
resulted in high metastasis potential and altered several functions
such as cell migration and invasion in vitro (Fig. 5 and 6A). These
observations suggested that HYAL1 plays a role in promoting the
invasive potential of breast cancer cells. It might also be a marker
predicting subsequent development of invasive breast cancer.
One of the well-studied functions of the HA and HAase system
is the generation of angiogenic HA fragments . These
angiogenic HA fragments have been shown to induce endothelial
cell proliferation, migration, and adhesion [26,31]. The secretion
of HAase by tumor cells has been shown to induce angiogenesis
. Angiogenic HA fragments are present in the urine of grade 2
and 3 bladder cancer patients, suggesting that the HA and HYAL1
system is active in bladder cancer . The HYAL1 and HYAL2
are widely distributed and degrade high MW HA in collaboration
with CD44 . We previously demonstrated that knockdown of
HYAL1 expression in breast cancer cells resulted in decreased
angiogenesis . In this study, we showed that upregulation of
HYAL1 expression induced higher angiogenesis in vitro and in
vivo (Fig. 6B and 7D). This was in accordance with previous
reports that HYAL1 over-expression increased MVD in rat colon
carcinoma xenografts , as well as the correlation of HYAL1
with MVD in bladder tumor . Which suggests that HYAL1
promotes tumor angiogenesis might be a general effect. Further
studies characterizing this in other cancer models would be
At present, whether HAase is a tumor promoter or a repressor
has been controversial. The results presented in this study showed
that forcing HYAL1 expression promoted tumor growth, invasion
and angiogenesis supporting its role as a tumor promoter. HYAL1
levels in various cancers were associated with high-grade invasive
tumors [9–10,20,27]. However, Jacobson et al.  found that the
overexpression of HYAL1 by cDNA transfection in a rat colon
carcinoma line decreased tumor growth, although the tumors were
Figure 6. Upregulation of HYAL1 in breast cancer cells promoted cell invasion and angiogenic capability in vitro. Invasion assays (A)
were performed using ECM gel-coated transwell culture chambers. 48 h after seeding for both MCF7 and ZR-75-30, the number of invasive cells was
increased in pcDNA3.1-HYAL1 transfectants (p,0.01). Angiogenesis assay (B) for CRL-1730 cells formed branch-like or net-like structures through co-
culture with MCF7 and ZR-75-30 cells. After 96 h co-culture, the number of vessels developed by CRL-1730 cells was enhanced in pcDNA3.1-HYAL1
transfectants (p,0.05), (original magnification 6100).
HYAL1 and Breast Cancer
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angiogenic. HYAL1 and HYAL2 have been identified to inhibit
lung and renal carcinoma cell growth in vivo but not in vitro .
Nykopp TK, et al found that HYAL1 and HYAL2 were
coexpressed and significantly downregulated in endometrioid
endometrial cancer and correlated with the accumulation of HA
. The controversy surrounding HAase as a tumor promoter or
a suppressor was recently explained by Lokeshwar et al [1,21].
Selection of cells for expression of different HYAL1 levels showed
that cells expressing amounts found in tumor tissues and cells
promote tumor growth, invasion and angiogenesis. In contrast,
cells with HAase levels exceeding 100 milliunits/106cells, (i.e.;
levels that are not naturally expressed by tumor cells) exhibit
reduced tumor incidence and growth due to induction of
apoptosis. Therefore, the function of HAase as a tumor promoter
or a suppressor is a concentration dependent phenomenon and
levels in genitourinary tumors are consistent with tumor cell
derived HAase acting mainly as a tumor promoter.
It is also noteworthy that other proteins related to HA synthase
(HAS) and HA-receptor CD44 and RHAMM are also involved in
tumor growth and metastasis. For example, overexpression of
HAS2, HYAL2 and CD44 is implicated in the invasiveness of
breast cancer . Blocking HAS3 expression in prostate cancer
cells decreased cell growth in vitro and tumor growth in vivo .
Silencing of HAS2 suppressed the malignant phenotype of invasive
breast cancer cells . HAS2 expression induced mesenchymal
and transformed properties in normal epithelial cells, but
interestingly, HAS2 expression in the absence of HAase decreased
tumor growth in glioma cells. Moreover, interaction between
RHAMM and HA fragments was known to induce the mitogen-
activated proteinkinase pathway,
RHAMM was a useful prognostic indicator for breast cancer
. Down-regulated CD44 and HA synthase while upregulating
the HAases, suggested that dynamic feedback signalling and
complex mechanisms occur in the net deposition of HA .
These results showed that the HAS-HA-HAase system is involved
in the regulation of tumor growth and invasion.
Summarizing the observations by us and others, we favor the
hypothesis that HYAL1 may play a critical role in the longevity of
Figure 7. Upregulation of HYAL1 increased tumor growth, angiogenesis, HAase activity and HA expression of MCF7 cells in vivo.
Representative mouse bearing tumors, the average tumor volume (A) from 5thweeks and tumor weight (B) in MCF7-HYAL1 group were increased
significantly than in MCF7-Vec group (p,0.01). Western blot (C) and immunohistochemistry (D) analysis showed that the expression of HYAL1 protein
was enhanced in the MCF7-HYAL1 group obviously, but HYAL2 levels were not altered. Compared with MCF7-Vec, expression of ki67 and CD31 were
increased in MCF7-HYAL1 (D). ELISA-like assay measured HAase activity (E) and HA levels (F) present in the tissue extracts, the HAase activity and HA
levels in MCF7-HYAL1 were higher than MCF7-Vec (p,0.01, p,0.05, respectively).
HYAL1 and Breast Cancer
PLoS ONE | www.plosone.org6 July 2011 | Volume 6 | Issue 7 | e22836
a wide spectrum of breast cancer cells. In our study, upregulation
of HYAL1 promoted the cell growth, migration, invasion and
angiogenesis. Interestingly, forcing HYAL1 expression induced
stoma cells of tumor to secrete HA in vivo, although HYAL1 could
cleave HA. To date the expression pattern and function of the
HYAL1 gene in human tumors are not completely elucidated. As
to the mechanism of how HAS-HA-HAase system influences the
biology characteristics of human breast cancer cells, more
investigations will be accomplished in the future.
Materials and Methods
Cell lines and cell culture
The human breast cancer cell lines MCF7 and ZR-75-30,
mouse embryonal fibroblast cell line HIH-3T3 and human
umbilical vein endothelial cell line CRL-1730 were acquired from
the cell bank of Shanghai Institute of Biological Sciences, Chinese
Academy of Sciences. These cells were maintained with
RPMI1640 medium (Gibco BRL), supplemented with 10% (v/v)
fetal bovine serum (FBS) (Gibco BRL). The medium was replaced
every 2 d, cells were passaged every 5 to 6 d, checked routinely
and cultivated in a 5% (v/v) CO2incubator at 37uC.
Plasmid construction and transfection
Total RNA was extracted from breast cancer cells using a RNA
extraction kit (Invitrogen). Total RNA (,1 mg) was subjected to
first strand cDNA synthesis using a SuperscriptTMpre-amplifica-
tion system and oligo(dT) primers (Invitrogen). The PCR primers
for the entire coding region of HYAL1 (611-1918) designed with
HYAL1 cDNA sequence (Genebank: U96078), the forward
primer was 59-GAGAAGCTTGCCGCCATGGCAGCCCACC-
TGCTTCCC-39, the reverse primer was 59-CAATTGTCACCA-
CATGCTCTTCCGCTCACACCA-39. PCR conditions were as
follows: 4 min for pre-denaturation at 94uC, then 94uC for 15 s,
55uC for 15 s and 72uC for 30 s for 30 cycles, and a final extension
at 72uC for 10 min. The PCR product (1300 bp) was purified and
restrictively digested with Hind III and Mfe I, the plasmid
pcDNA3.1(+) (Invitrogen) was purified and restrictively digested
with Hind III and EcoR I, then cloned the PCR product into
pcDNA3.1 to form pcDNA3.1-HYAL1, the pcDNA3.1 vector was
treated as a control.
For transfection, MCF7 and ZR-75-30 were seeded in six-well
plates at 56105/well and incubated in 5% CO295% air incubator
at 37uC. When cells were ,70% confluence, cells were transfected
with Lipofectamine 2000 (Invitrogen) transfection reagent accord-
ing to the manufacturer’s protocol. Recombinant plasmid (4 mg)
were mixed with Lipofectamine and pre-incubated for 20 min at
room temperature in serum-free and antibiotic-free RPMI1640.
MCF7 cells transfected with pcDNA3.1-HYAL1 and pcDNA3.1
vector were named as MCF7-HYAL1 and MCF7-Vec, ZR-75-30
cells transfected with pcDNA3.1-HYAL1 and pcDNA3.1 vector
were named as ZR-HYAL1 and ZR-Vec, respectively. G418
(500 mg/ml) was applied to stable screeing and isolating the
resistant colonies. At the same time, corresponding empty vector
was transfected as control.
Semi-quantitative reverse transcription-polymerase chain
Total RNA extraction was same as before, following manufac-
turer’s instructions. The reverse transcription was carried out with
a SuperScript first-strand synthesis system (Invitrogen) using
Oligo(dT)12-18 primers. cDNA was amplified by Taq DNA
polymerase (Promega, USA). Human b-actin gene was used as
an internal control. Each PCR program involved a initial step
denaturation 5 min at 94uC, followed by 28 cycles (for HYAL1,
HYAL2 and b-actin) at 94uC for 20 s, 62uC for 20 s and 72uC for
30 s, at last 72uC for 10 min. DNA primer sequences were
designed as follows: for human HYAL1 gene (Accession No.
U96078), Sense: 59-TGGATGGCAGGCACCCTCCA-39, and
antisense strand: 59-CACCAGCAGCCACAGCCACA-39, the
amplicon size is 289 bp. For human HYAL2 gene (Accession
No. U09577), Sense: 59-TGGCCCGCAATGACCAGCTG-39,
and antisense strand: 59-GCCGCACTCTCGCCAATGGT-39,
the amplicon size is 262 bp. For b-actin gene (accession No.
reverse primer, 59-GGGGTGTTGAAGGTCTCAAA-39, the
amplicon size was 139 bp. The PCR amplified products were
separated by 1.5% agarose gels electrophoresis, and the bands
were visualized by staining with 0.5% Goldview. Gel images were
obtained and the densities of PCR products were quantified by
using densitometry methods.
Western blot analysis
The cells and fresh tissue specimens (0.5–1 g) were harvested
and total protein was extracted with RIPA buffer containing
protease inhibitors, protein concentration was determined by the
Bradford assay (Bio-Rad Laboratories, Hercules, CA). Equal
amounts of protein were subjected to 7.5% SDS–polyacrylamide
gel and the resolved proteins were transferred electrophoretically
to PVDF membranes (Millipore, Bedford, MA, USA). Membranes
were blocked for 1 h with 5% non-fat milk in PBST buffer at room
temperature, then were incubated with antibodies against HYAL1
(rabbit polyclonal; 1:500; Sigma, USA), HYAL2 (rabbit polyclon-
al; 1:500; Abcam, UK), and b-actin (rabbit polyclonal; 1:1000;
Santa Cruz, CA) for overnight at 4uC, respectively. The
membranes were washed for three times with PBST, and then
were incubated with the respective secondary antibodies for 1 h at
room temperature. Membranes were incubated with enhanced
chemiluminescence (ECL) (Pierce, Rockford, IL, USA), exposed to
X-ray film for 1–2 min. The results were analyzed by using the
Quantity One 4.6.3 software (Bio-Rad, Hercules, CA).
The transfected cells were cultured on sterile cover slips for 24 h
and washed twice with cool PBS. Cells were then fixed with 2%
formaldehyde, permeabilized with 0.1% Triton X-100, blocked
with 2% BSA for 30 min at room temperature, and incubated
with the primary antibody overnight at 4uC. Samples were
washed, incubated with the secondary antibody for 30 min.
Immunofluorescence was analysed using a confocal microscope.
Soft agar growth assay
(0.56105cells/well in 6-well plate) were suspended in 3 ml
RPMI1640 containing 10% FBS (pre-warmed to 37uC), and
300 ml of 3% agarose in PBS (pre-warmed to 60uC) was added.
Agar suspended cells (1 ml/well in 6-well plates) were plated out in
dishes coated with 1 ml of agar-coated dishes (0.6% agarose in
RPMI1640). After solidification at room temperature for 20 min,
3 ml complete medium was added to each well and cells were
incubated at 37uC in a humidified atmosphere of 5% CO2(v/v) in
air for 2 weeks. After this period, 10 fields were randomly selected
and the numbers of colony were counted under a microscope.
or pcDNA3.1-HYAL1transfected cells
3D Matrigel culture
Matrigel basement membrane matrix (BD Biosciences, San
Jose, CA) was thawed on ice and coated onto 24-wells cluster
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plates as a bottom layer. Subsequently, a total of 5,000 cells in
100 ml growth medium resuspended with 100 ml Matrigel was
seeded on top of this bottom layer. Growth medium (0.5 ml) was
added on top of the Matrigel after the polymerization was
completed. Experiments were performed in triplicate. After 10
days, colonies were counted and colony sizes were measured under
the stereomicroscope at 1006magnification.
MTT proliferation assay
The capability of cell proliferation was measured by [3-(4, 5-
dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide] MTT
assay. Briefly, cells were plated at 56103cells/well in 96-well
plates and incubated for 0, 2, 4, 6, 8 and 10 d, respectively. Then
cells were incubated with 20 ml MTT (10 mg/ml) for 4 h at 37uC
and 100 ml DMSO (Sigma Chemical Co., USA) was pipetted to
solubilize the crystal product for 10 min at room temperature. The
absorbance (A) of each well was measured with a microplate
reader (Bio-Rad) at a wavelength of 490 nm. This experiment was
repeated three times.
ELISA-like assay for HAase activity
HAase activity in serum-free conditioned medium of transfec-
tants was assayed using the HAase ELISA-like assay as described
previously . Fresh tissue specimens (,0.5–l.0 g) were sus-
pended in an ice-cold homogenization buffer (5 mM Hepes
pH 7.2 and 1 mM benzamidine-HCl) and homogenized for 30 s
using the tissue homogenizer. The tissue extracts were clarified by
centrifugation at 10,000 rpm for 30 min. The extracts were
assayed for protein concentration, HAase activity and HA
ELISA-like assay for HA
The HA ELISA-like assay was described previously .
Briefly, ELISA plates coated with HA binding protein were
incubated with samples or standards (1 h, room temperature) in
triplicate, washed four times with washing buffer, incubated with a
solution containing horseradish peroxidase-conjugated HA-bind-
ing protein (30 min, room temperature), washed again four times,
and incubated with 100 ml of the substrate solution in the dark at
room temperature. After 30 min, the reaction was stopped by
adding 50 ml stop solution to each well, and the absorbance was
measured at a wavelength of 405 nm.
Flow cytometry assay
Different cell cycle phases (G0/G1, S and G2/M phase) are
characterized by different DNA contents. The control, pcDNA3.1-
Vec and pcDNA3.1-HYAL1 transfected cells were harvested with
trypsin-EDTA, washed with chilled PBS twice and fixed with 70%
ethanol at 220uC for 2 h, respectively. The fixed cells were
pelleted, re-suspended in PI/RNase/PBS (100 mg/ml propidium
iodide and 10 mg/ml RNase A) for at least 30 min at 37uC in dark.
Cell cycle analysis was performed on flow cytometer. This
experiment was repeated three times.
Wound healing assay
Cells were cultured in standard conditions, as described above.
Until 100% confluence, the migration potency was determined
using scratch wound healing assay. The scratched plates were
photographed at the center of the wells using a standard
magnification of 1006. The scratched cells were maintained
under standard conditions for 24 hr and 48 hr. Plates were
washed once again, and then the plates were photographed at the
same sites of the wells using the same magnification. The cells
migrating into the scratched area from the wound edge per picture
were counted. The impact of pcDNA3.1-HYAL1 on cell
migration potency was evaluated by comparing the mean of
migration width with pcDNA3.1 empty vector.
Cell invasion assay
Invasion capability in vitro was measured in transwell chambers
(Costar Inc, USA) according to the protocol of the manufacturer.
Briefly, the upper chambers of the transwell inserts were coated
with 100 ml diluted ECM gel solution at 37uC for 4 h, and then
pretreated with serum-free RPMI1640 medium at 37uC for 1 h
before seeding the breast cancer cells at a density of 16105/well in
100 ml medium with 1% FBS. The lower chambers were filled
with 500 ml RPMI1640 with 10% FBS and HIH-3T3 contained
medium as a chemoattractant. The transwells were then incubated
at 37uC with 5% CO2for 48 h to allow cells to migrate. At the end
of incubation, the cells on the upper side of the insert filter were
completely removed by wiping with cotton swab, and the cells that
had invaded through the ECM gel-coated filter were fixed in
ethanol and stained with H&E. For quantification, the cells were
counted under a microscope on 5 random fields at 1006. This
experiment was repeated three times.
Human umbilical vein endothelial cell line CRL-1730 were
allowed to grow in transwell chambers coated with ECM gel as
described above for the invasion assay (26105cells/chamber). At
the same time, the breast cancer cells were plated into 6-well plates
(26105cells/well), and cultured overnight. Then the transwell
chambers were set in the 6-well plates where tumor cells were
already added. The cells were co-cultured for 96 h, and the
medium was changed every 24 h. The transwell chambers were
washed three times with PBS, fixed in ethanol and stained with
H&E. The membranes were carefully taken out of the chamber,
set on glass slides, covered with a coverslip, observed under
microscope, and documented with a digital imaging system (Leica
MD20, Germany). The degree of angiogenesis was measured by
the following method: number of branch points and the total
number of branches per point, with the product indicating the
degree of angiogenesis. This experiment was repeated three times.
In vivo assay
Balb/c nude mice were purchased from Vital River Lab Animal
Technology Co. Ltd, and maintained under specific-pathogen-free
conditions in Experimental Animal Department of the Chongqing
Medical University (Chongqing, China). The protocols were
approved by the Institutional Animal Care and Use Committee in
Chongqing Medical University (CQMU-2008-127). Mice were
randomly divided into 2 groups (MCF7-Vec and MCF7-HYAL1)
with 10 mice in each group. MCF7 cells at 16107cells per l00 ml
of serum-free medium were injected orthotopically into the
mammary fat-pad of 6 weeks old female Balb/C nude mice.
Tumor diameters were measured one time per week with a
caliper, and the volume of tumors were calculated by the following
formula: x 6 y2/2, where x is the largest diameter of the tumor
and y is the shortest diameter. At 9thweek after the injection, the
tumors were harvested for western blot, immunohistochmistry,
HAase activity and HA levels analysis.
The tissue sections (,5 mm) were de-paraffinized in xylene,
rehydrated in graded ethanol solutions, permeabilized in 0.1%
Triton X-100 and 0.1% sodium citrate. The endogenous
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peroxidase activity was quenched by incubation of the sections in
0.3% hydrogen peroxide with methanol. Subsequently, the slides
were treated with 1% bovine serum albumin for 30 min to reduce
nonspecific binding, followed by incubation overnight with
antibody against HYAL1 (rabbit polyclonal; Sigma, USA),
HYAL2 (rabbit polyclonal; Abcam, UK), Ki67 (rabbit polyclonal;
Abcam, UK) and CD31 (rabbit polyclonal; Abcam, UK) at a
dilution of 1:200, respectively. After washing, the slides were
further incubated with HRP-conjugated secondary antibody
followed by 3,3-diaminobenzidine solution at 4uC for 30 min.
Hematoxylin was used for nucleus counterstaining. For negative
controls, the antibody was replaced by normal goat serum. Slides
were photographed with microscope attached with CCD camera.
Results are represented as means 6 standard deviation (SD).
Paired and 2-tailed Student’s t-tests were used to compare results
from the experiments. A p-value of less than 0.05 was considered
We thank Dr Qi Cai for critical reading of the manuscript, and we would
like to thank all our colleagues and collaborators who have participated in
Conceived and designed the experiments: JXT GSR. Performed the
experiments: JXT XYW XLS HYL YS LW. Analyzed the data: JXT
XYW XLS HYL GSR. Contributed reagents/materials/analysis tools:
JXT XYW YS LW. Wrote the paper: JXT GSR.
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