Human hyaluronic acid synthase-1 promotes malignant transformation via epithelial-to-mesenchymal transition, micronucleation and centrosome abnormalities
Nguyen et al. Cell Communication and Signaling
Human hyaluronic acid synthase-1 promotes malignant transformation via epithelial-to-mesenchymal transition, micronucleation and centrosome abnormalities
Nguyet Nguyen 0 2
Awanit Kumar 0 2
Simi Chacko 0 2
Rodney J. Ouellette 1 2
Anirban Ghosh 1 2
0 Equal contributors
1 Department of Chemistry and Biochemistry, Université de Moncton , Moncton, NB , Canada
2 Atlantic Cancer Research Institute , 35 Providence Street, Moncton, NB E1C 8X3 , Canada
Background: Human hyaluronic acid (HA) molecules are synthesized by three membrane spanning Hyaluronic Acid Synthases (HAS1, HAS2 and HAS3). Of the three, HAS1 is found to be localized more into the cytoplasmic space where it synthesizes intracellular HA. HA is a ubiquitous glycosaminoglycan, mainly present in the extracellular matrix (ECM) and on the cell surface, but are also detected intracellularly. Accumulation of HA in cancer cells, the cancer-surrounding stroma, and ECM is generally considered an independent prognostic factors for patients. Higher HA production also correlates with higher tumor grade and more genetic heterogeneity in multiple cancer types which is known to contribute to drug resistance and results in treatment failure. Tumor heterogeneity and intra-tumor clonal diversity are major challenges for diagnosis and treatment. Identification of the driver pathway(s) that initiate genomic instability, tumor heterogeneity and subsequent phenotypic/clinical manifestations, are fundamental for the diagnosis and treatment of cancer. Thus far, no evidence was shown to correlate intracellular HA status (produced by HAS1) and the generation of genetic diversity in tumors. Methods: We tested different cell lines engineered to induce HAS1 expression. We measured the epithelial traits, centrosomal abnormalities, micronucleation and polynucleation of those HAS1-expressing cells. We performed real-time PCR, 3D cell culture assay, confocal microscopy, immunoblots and HA-capture methods. Results: Our results demonstrate that overexpression of HAS1 induces loss of epithelial traits, increases centrosomal abnormalities, micronucleation and polynucleation, which together indicate manifestation of malignant transformation, intratumoral genetic heterogeneity, and possibly create suitable niche for cancer stem cells generation. Conclusions: The intracellular HA produced by HAS1 can aggravate genomic instability and intratumor heterogeneity, pointing to a fundamental role of intracellular HA in cancer initiation and progression.
Hyaluronic acid Synthase-1; Malignant transformation; Epithelial-to-Mesenchymal transition; Genetic heterogeneity; Genomic instability; Micronucleus; Chromosomal instability and Centrosome abnormalities
Hyaluronan or hyaluronic acid (HA) is a ubiquitous
glycosaminoglycan mainly present in the extracellular
matrix (ECM) and on the cell surface, but also present
intracellularly where it is often associated with nucleoli
and nuclear clefts [
]. HA is composed of repeating
disaccharide units (N-acetylglucosamine and D-glucuronic
acid), and in human HA molecules are synthesized by
three Hyaluronic Acid Synthases [HAS1 (hCh19), HAS2
(hCh8), and HAS3 (hCh16)], each of which contains
multiple transmembrane domains. Aberrant endogenous
production of HA or treatment with exogenous HA in
vitro has also been shown to promote cancer cell growth
and malignant behavior in multiple model systems [
Overproduction of HA in breast carcinoma cells and in
the surrounding stroma are considered independent
prognostic factors for patient survival and correlate with higher
tumor grade [
]. In addition, elevated serum HA levels are
a hallmark of metastatic breast cancer [
Most of the studies that correlate overexpressed HA in
cancer have been focused on HAS2 and HAS3 [
Among three isoenzymes, HAS2 and HAS3 have been
shown to produce extracellular HA, whereas HA
synthesized by HAS1 has been identified in both intra- and
extracellular compartments of cancer cells [
3, 9, 13
HAS1 is mostly localized in the cytoplasmic space
where it synthesizes intracellular HA  and appears to
require a larger amount of substrate (UDP-N-acetyl
] for the production of HA. The
production of UDP-N-acetyl glucosamine is increased in all
cancers due to observed dependence to the glycolytic
We and others have identified HAS1 as a key
contributor to oncogenesis and disease progression in both
hematological and solid cancers [
]. Most of
the previous studies on HA synthesized by HAS2 and
HAS3 were focused mainly on its roles in the ECM and
signal transduction, whereas the majority of HAS1
research has focused on prognostic marker studies or
expression profiles of HAS1 genes in different cancers
]. In a recent retrospective histological study,
authors found that a less favorable outcome of breast
carcinoma patients is strongly associated with HAS1
expression (but not HAS2 and HAS3) as seen with
shorter overall survival, higher relapse rate, estrogen
receptor negativity and HER2 positivity . In this
study HAS1 was found to localize mostly in cytoplasm.
HAS1 is also implicated in the growth and development
of breast cancers, as well as the generation of
intratumor heterogeneity [
] that maintains a cancer stem
cell-like trait or phenotype [
]. HAS1 has been shown
to be prognostic factor in multiple myeloma [
] and bladder cancer [
] and is overexpressed
in a variety of other cancers [
Breast cancer and most other solid tumors display
substantial cellular and genetic heterogeneity [
which are used to establish clinical grade. Centrosome
abnormalities and micronucleation are the prominent
histological phenotypes of human cancers, including
breast carcinoma [
]. Decades of histopathological
observations lead to the hypothesis that centrosome
abnormalities result in chromosomal instability (CIN)
and that they have progressive involvement in advanced
stages of carcinogenesis [
So far polysaccharide synthesis has not been
mechanistically or molecularly correlated to causalities of
carcinogenesis. Although ever-growing evidence indicates that
the accumulation of intracellular and stromal HA during
mammary carcinogenesis plays a role in cancer
progression, a role for intracellular HA in genetic instability,
generation of clonal diversity and cellular transformation in
breast carcinoma or any other cancer, has not been
]. Thus, the identification of a specific driver
mechanism that generates centrosomal abnormality,
genetic instability, micro-nucleus formation is critical for
better understanding and treatment. Here we demonstrate
a correlation between intracellular HA synthesized by
HAS1 and the generation of clonal diversity and
prevalence of genetic instability in cells. The results of this
study suggests that overexpression of HAS1 induces loss
of epithelial traits, centrosomal abnormalities,
micronucleation and polynucleation, all of which are
manifestations of malignant transformation. These observations
reveal a previously unknown role of HAS1 in intracellular
HA-mediated cellular transformation.
Cells, reagents and plasmids
MCF10A, HeLa and DLD1 cells were originally procured
from the American Type Culture Collection (Manassas,
VA) and grown in recommended culture media and
conditions. MCF 10A, a non-tumorigenic mammary
epithelial cell line is one of the widely used model to study
loss of epithelial traits on 3D tissue culture. We chose
DLD1 (colorectal adenocarcinoma) cell line, as 86% of
population of these cells are diploid (46 chromosomes).
Also the different cell lines were used to show the effect
of HA overexpression in cell lines of diverse origin.
All the monoclonal antibodies were purchased from
SantaCruz Biotech or Calbiochem. Anti-CD44 (F4 and
DF1485), anti-BRCA1 (BR64 and D9), and GM130
(H7) antibodies were purchased from Santa Cruze
Biotech. Anti-GFP (MAB2510) was from Calbiochem, and
pericentrin antibody (ab4448–100) was from Abcam.
Anti-A2 (a monoclonal antibody against
A2-fusiontag) was a kind gift from Prof. Greg Matlashewski,
McGill University, Quebec, Canada). Transfection
reagent Lipofectamine 2000 and RNA isolation reagent
(TRIzol reagent) were from LifeTechnology.
Biotinylatedhyalurone-binding-protein (bHABP) was purchased from
Sigma. The mammalian HAS1 expression plasmids
(pCDNA3 and pCEP4) were used from the previous
published work [
]. Human HAS2 cDNA was purchased
from OriGene. HAS1, HAS2 and GFP cDNAs were
subcloned in pTRE2hyg or pTRE2pur vector from Clontech
Laboratories, Inc. for conditional expression (tetracycline
inducible). All the Tetracycline-inducible cell lines and
their controls were subcultured in tetracycline-free media
for maintenance. The tetracycline-on induction was done
with doxycycline (Dox) in tetracycline-free media. The cell
synchronization was done using double-thymidine-block
method as described elsewhere [
]. Briefly, HeLa cells at
30% confluency was treated with 2 mM thymidine (final
concentration) in culture media for 16 h, washed and
incubated with normal media for 9 h. This was followed by
a second thymidine treatment for 17 h. After second
thymidine treatment the cells were grown in normal
media for 10 more hours to collect for mitosis stage and
16 h to collect for G1/S stages.
Total RNA was harvested with the TRIzol followed by
RNeasy Kit (Qiagen) assisted isolation. One microgram
of total RNA was used with the QuantiTect Reverse
Transcription Kit (Qiagen) to synthesize cDNA. qPCR
was performed using B-R SYBR Green SuperMix from
iQuanta Biosciences using an Eppendorf Realplex2
Mastercycler. The ΔΔCt was calculated to identify fold
change in gene expression normalized to GAPDH.
Primers for E-Cadherin (5′-ATGCTGAGGATGGAGG
TGGGT and 5′-CAAATGTGTTCAGCTCAGCCAG
CA), N-Cadherin (5′-TGTGGGAATCCGACGAATGG
ATGA and 5′-TGGAGCCACTGCCTTCATAGTCAA),
and GAPDH (5′-ACAGTCAGCCGCATCTTCTT and 5′
ACGACCAAATCCGTTGACTC) were used for qPCR.
Transfection, selection and expression
The plasmid constructs were transfected to cells using
Lipofectamine 2000 in 6-well tissue culture plates
following manufacturer’s instructions. The cells were
selected using appropriate antibiotic in tetracycline free
media for two or 3 weeks followed by cultured into low
antibiotic concentrations to maintain in tetracycline
free media or frozen as cell-line-stock. We followed
Clontech Laboratories manuals for TET-On expression
induction system (pTRE2). In brief, for conditional
expression of cDNA under
tetracycline-responsiveelements, cells were first transfected and selected with
pTET-On vector. The resulted populations were further
transfected and selected with HAS1, HAS2 or GFP
subcloned in pTRE2hyg and/or pTRE2pur for induced
Immunofluorescence microscopy and HA capture of active HAS1
For most of the immunofluorescence (IF) experiments
cells were seeded onto 8-well chamber slides. The
details of the IF methods were followed from our previous
]. Briefly, the cells were fixed,
permeabilized and blocked followed by overnight incubation
with primary antibody or bHABP at 4 °C for protein or
HA staining respectively. This was followed by
incubation with secondary antibody and/or phalloidin
conjugated to Alexa Fluor 594 Dyes (Molecular Probes,
Invitrogen, Eugene, Oregon, USA) or streptavidin
conjugated to Alexa Fluor 350 Dyes (Molecular Probes,
Invitrogen, Eugene, Oregon, USA) in blocking solution
for 1–2 h at room temperature, and finally mounted
with PermaFluor aqueous mountant (Thermo Fisher
Scientific, UK). We used Olympus FV1000 confocal
microscope and used their image capture and analytical
software (ASW2.1) for z-stacking, nuclear perimeter and
fluorescent measurements for quantitation. For the
capture of HA from actively synthesizing HAS1 and other
HA-binding proteins, cell lysate were subjected to bHABP
mediated HA-capture following the method described in
our previous publication [
]. Briefly, cleared cell lysates
were incubated with bHABP followed by incubation with
streptavidin-sepharose beads (GE Healthcare) to collect
HA bound to bHABP.
3D reconstituted cell-culture
A 3-dimensional cultures system was adopted where cells
were seeded on top of the Matrigel. Matrigel™ Basement
Membrane Matrix from BD Biosciences was used. Bottom
of the cell culture plates (48-well) was layered with 50%
Matrigel in MCF10A growth media and let them solidified
as basement matrix. On that matrix 10,000 cells per well
in 2% Matrigel with media (0.5 ml) were incubated. Twice
every week half of the media was replaced with fresh
media with 2% Matrigel. Cultures were monitored over 2
weeks for development of 3-dimensional ‘acini’ structures
Cell growth assay
Cell growth was monitored using the CellTiter-Blue® Cell
Viability Assay Kit (Promega). Briefly, 5000 cells per well of
96 well plates were seeded in triplicate, and separate
plates were used for each day of measurement. Different
concentration of Doxycycline was added for induction of
Tet-responsive genes as indicated. For measurements
CellTiter-Blue® substrate was added, incubated for 1 h at
37 °C in 5% CO2 and fluorescence recorded at 560Ex/
590Em. The fluorescence readings were normalized to day
0 of vehicle-control group and plotted as fold increased.
Overexpression of human HAS1 increases intracellular HA
The normal mammary cell line MCF10A was
transfected with plasmids that express HAS1 or an unrelated
protozoan (Leishmania major) gene (LMA2) and were
selected using appropriate antibiotic for 2 weeks.
Leishmania major gene Lm2415 with A2 fusion tag
(LMA2) has no homology with any mammalian gene,
and hence we used as a control gene. It had the same
A2 fusion-tag which was used to identify HAS1
expression as recombinant protein [
]. The selected
populations of MCF10A cells were then seeded onto 8-well
chamber slides, grown for 40 h and subjected to HA
staining using biotinylated bovine HA binding protein
(bHABP) and streptavidin-Alexa-488 fluorescent probes.
MCF10A cells that express HAS1 with different
mammalian expression plasmid backbones and fusion tags showed
similar cytoplasmic localization of HA and significantly
more HA staining in comparison to protozoa-gene
transfected MCF10A (Fig. 1a). The punctate localization of the
HA synthesized by overexpressed HAS1 inside the cell
were observed in agreement with previously published
1, 2, 13
]. We also transiently transfected HAS1 into
primary lung cells and observed cytoplasmic localization
of synthesized HA (Additional file 1: Figure S1A). A
similar pattern of intracellular HA expression was also
observed in HeLa cells (Additional file 1: Figure S1B) and
in DLD1 cells (Additional file 1: Figure S1C) with a
tetracycline-inducible system of HAS1 expression, where
the cells were transfected and selected for inducible cDNA
expression: HAS1 in pTRE2-vector (puromycin). These
data indicate that overexpression of HAS1 causes an
increase in the expression of intracellular HA. We also
evaluated whether the overexpression of HAS1 has any effect
of cellular growth. We observed a lower mitotic index
(Additional file 1: Figure S2A) for MCF10A cells that
express HAS1 in comparison to LMA2-expressing or mock
transfected MCF10A cells. Mitotic index is percentage of
cells undergoing mitosis per 100 non-mitotic cells, and is
a measure of the cellular growth rate. Similarly, as shown
in the Additional file 2: Figure S2B, HeLa cells with
tetracycline-inducible HAS1, non-induced background
expression with 0 μg/ml Dox as well as 1, 3 and 6 μg/ml
Dox inductions slowed the cell-population growth
compared to the similar induction-scale of
tetracyclineinducible GFP expressing cells. The higher the induction
of the HAS1, the slower was the growth, with cells ceasing
to grow at 6 μg/ml Dox induction on day 13 of culturing.
CD44 and its splice-variants are well-characterized HA
receptors that are implicated in cellular transformation,
as well as being a stem cell marker [
therefore sought to determine whether overexpression of
HAS1 and the resulting increase in cytoplasmic HA is
related to concomitant increase in cytoplasmic CD44
expression and/or localization. To answer this we
transfected MCF10A cells, selected and seeded onto 8-well
chamber slides for HA- and immuno- staining. As shown
in the Fig. 1b the expression of CD44 in the cytoplasm
was significantly higher in the HAS1 overexpressing cells
than in the control LMA2-expressing MCF10A cells, and
HA and CD44 were found to be co-localized in many
areas of the cell. These results indicate that cytoplasmic
overexpression of HA, synthesized by HAS1,
concomitantly increased the endogenous expression of CD44 in
Overexpression of HAS1 induces epithelial-to
Mesenchymal transition (EMT) in MCF10A cells
EMT is one of the hallmarks of cellular invasiveness /
transformation and widely regarded as phenotype of
cancer progression. As HAS1 is a prominent prognostic
factor in breast cancer and other cancers [
sought to determine whether overexpression of HAS1
influences EMT and thereby skew the cellular fate. MCF10A
cells grown in 3D culture produce mammary epithelial
acini-mimicking structures; however, in contrast the
induction of EMT in MCF10A cells causes a diffuse network
of cells without any 3D structure . MCF10A cells were
mock transfected (no plasmid) or transfected with a
plasmid that expresses HAS1 or an unrelated protozoa gene
(LMA2). The selected cell populations were subjected to
reconstituted basement membrane 3D culture using
Matrigel. Mock-transfected or protozoa-gene-transfected
MCF10A cultures developed acini structures characteristic
of the epithelial nature of the cells (Fig. 2a) whereas
MCF10A cell-population selected with HAS1
distinctively did not produce any acini structures, but rather
showed a loose cellular network, characteristic of
transformed mesenchymal-type cells. Figure 2a is
representative of four independent experiments. To identify
EMT gene-expression signatures we performed
quantitative RT-PCR analysis of E-cadherin (epithelial) and
Ncadherin (mesenchymal) in HAS1-expressing cells as well
as control populations. Quantification of E-cadherin
and N-cadherin transcripts was performed using
RTqPCR and GAPDH (Glyceraldehyde 3-phosphate
dehydrogenase) was used as normalization control for
ΔΔCt measurements. The quantitation of the fold
increase was measured by 2-ΔΔCt (where ΔΔCt = ΔCt of
sample - ΔCt of GAPDH). Relative expression of
Ecadherin transcript was significantly diminished in
HAS1expressing cells (p = 0.005) whereas the expression of
N-cadherin increased by almost 10-fold in
HAS1expresing cells (p = 0.024), suggesting that MCF10A
cells that overexpressed HAS1 induced EMT (Fig. 2b).
The data is representative of three experiments
(triplicate per sample per experiment).
Prolonged HAS1 expression induces clonal diversity in cell-population
Intra-tumor clonal diversity as a measure of cancer
progression and drug resistance is well studied [
However, correlation of clonal diversity (and
aneuploidy) induced by polysaccharide synthesis has not
Fig. 2 Overexpression of HAS1 induces Epithelial-to-Mesenchymal Transition (EMT) in MCF10A cells. a The normal mammary cell line MCF10A
was mock transfected (left), or transfected with a plasmids that express HAS1 (middle) or an unrelated protozoa gene LMA2 (right). The selected
populations were cultured in 3D reconstituted basement-membrane model. Both control panels (right and left) show acini structure in contrast
to middle panel. b MCF10A cells were transfected as described in (a) and subjected to EMT analysis using the common EMT markers E-cadherin
(Epithelial) and N-cadherin (Mesenchymal) using RT-qPCR. The relative expression of E-cadherin is diminished, however N-cadherin is over-expressed
in MCF10A-HAS1 cells
MCF10A-MOCK MCF10A-HAS1 MCF10A-LMA2
been described. We observed these strikingly unique
effects in the progeny cell populations that ectopically
express HAS1 for several generations. Transient
overexpression of HAS1 for 40 to 72 h did not produce a
cell population with differing cellular and nuclear
morphologies (data not shown) [
]. As shown in Fig.
3a, a diverse and heterogeneous population emerged
when HeLa cells were induced (+Dox) to express HAS1
for a prolonged period (10 weeks, 3 μg/ml Dox),
however the cells were morphologically similar to each
other in the non-induced (−Dox) population. These
cells were also engineered to co-express GFP under
tetracycline responsive promoters to verify conditional
expression of GFP due to induction with Dox and to
identify any background (leaky) expression of this
tetracycline-inducible plasmid system without any Dox
induction. Similarly we also observed high population
diversity in nuclear morphology (Fig. 3b) when HAS1
was expressed under a CMV promoter continuously for
10 weeks after transfection and selection in MCF10A
cells compared to mock-transfected cells that express
an unrelated protozoa gene (LMA2).
We also tested DLD1 cells (colorectal adenocarcinoma
cell line, 46 chromosomes occurring in 86% of
population) for the effects of HAS1 overexpression using a
tetracycline-inducible system and observed similar
outcome of emergent population’s diversity (shown as nuclear
perimeter) when HAS1 was overexpressed (4 weeks
induction with 3 μg/ml of Dox), but no effects on nuclear
morphology in control cells (pTET) or cells that
overexpressed the related HAS2 (Fig. 3c). These results indicate
that expression of cytoplasmic HA synthesized by HAS1
induces population diversity irrespective of cell types and
HAS1 expression induces micronucleus formation
Cytogenetically the progression of cancer is driven by
the generation of population diversity, micronucleus
formation and chromosomal instability which generate
aggressive clones, drug-resistance phenotypes, and the
emergence of cancer stem cells [
]. We observed
that long-term HAS1-expressing cells produce
morphologically divergent cells with an abnormally high
incidence of micronucleated and polynucleated cells,
possibly indicating ongoing generation of aneuploidy and
continued chromosomal instability. As demonstrated in
Fig. 4a, the mock- and LMA2-transfected and selected
(6 to 7 weeks after transfection) population of MCF10A
cells have 5–10 μ-nuclei per 100 nuclei, whereas in
HAS1-transfected cells have 30 to 50 micronuclei per
DLD1 pTET HAS1 HAS2
100 nuclei. We further verified the above
HAS1associated phenomenon in tetracycline-inducible DLD1
cells, which conditionally expressed HAS1 and HAS2.
Expression of HAS1 and HAS2 was induced with Dox for
short-term (100 h, Fig. 4b) as well as long-term (4 weeks,
Fig. 4c) to examine micronucleus formation. As depicted
in Fig. 4b DLD1 cells reproduced the phenomenon of
micronuclei formation upon short-term HAS1 expression
(p = 0.078), but micronuelci were not observed in
HAS2expressing (p = 0.96) and control DLD1 (pTET) (p = 0.71)
cell populations. Cells that contain the inducible HAS1
plasmid showed a significant increase in micronuclei
formation over control and HAS2 cells in the absence of Dox
induction due to leaky HAS1 expression, corroborating
the fact that minimal HAS1 expression (but not HAS2)
induces micronuclei formation (Fig. 4b). Representative
photographs of short-term Dox induction is shown in
Fig. 4d. A similar pattern of micronuclei formation was
observed for long-term HAS1 induction (4 weeks) in
DLD1 cells (Fig. 4c). The rate of micronucleation did not
significantly differ in short- verses long-term induction of
HAS1, possibly indicating sustained chromosomal
instability once cytoplasmic HA levels were increased by
HAS1 expression compromises centrosome integrity
Mitotic aberrations are the result of many possible
mitotic abnormalities. Most cells with mitotic aberrations
die off from the population, but cancer cells inherently
sustain signatures of mitotic aberrations to generate clonal
diversity. One of the most predominant mitotic
aberrations is caused by centrosome abnormalities (amplification
by number and volume), which are correlated to cancer
]. Centrosome abnormalities and a high
incidence of micronuclei are considered hallmark
signatures of chromosomal instability and are found in all solid
tumors, including breast cancer [
]. Here we tested
whether cells with HAS1-synthesized cytoplasmic HA,
which resulted in clonal diversity and micronucleation,
could also display signatures of centrosomal
abnormalities. Immunofluorescence staining of centrosomes with
anti-pericentrin antibody revealed enlarged, fragmented
or multipolar centrosomes in HAS1-expressing MCF10A
populations but not in control LMA2-expressing cells
(Fig. 5a). Pericentrin is integral to the filamentous matrix
of the centrosome and is involved in the initial
establishment of organized microtubule arrays of the mitotic
apparatus. A similar pattern of centrosome abnormalities
were also observed in tetracycline-inducible
HAS1expressing HeLa and DLD1 cells, but not in control
(pTET) and HAS2-expressing populations (Fig. 5b). To
quantitate the enlargement of centrosomes (Fig. 5c) we
extracted fluorescence intensity from the region of interest
(RIO) of anti-pericentrin staining (red channel) from
multiple fields of confocal images collected from DLD1
cells. The ROI fluorescent units of pericentrin represent
the comparative volume and number of centrosomes,
which was significantly increased in HAS1-expressing
cells (Fig. 5c). We also observed a comparatively larger
Golgi apparatus when cells undergo HAS1-induced
centrosome abnormalities (Additional file 3: Figure
S3A). The above observations of centrosome
abnormalities caused by HAS1 expression in multiple cell types
confirm the association of HAS1 expression with
RHAMM and BRCA1 interact with HAS1-synthesized cytoplasmic HA
Hyaluronan-mediated motility receptor (RHAMM),
also known as CD168, is an HA-binding protein and
has been implicated in mitotic spindle
formation/stability, correlated with poor outcomes in many cancers
], and is implicated in metastasis [
results correlating micronucleus generation and
centrosome abnormalities with HAS1 expression prompted us
to investigate the possible molecular interactions between
spindle-centrosome assembly and cytoplasmic HA
produced by HAS1. The dynamics of proper mitotic spindle
machinery formation, stability and segregation relies on
many proteins (for example RHAMM) [
possesses partially overlapping centrosome-binding and
HA-binding domains [
], which may provide a potential
clue as to how cytoplasmic HA interferes with spindle
formation. Naturally occurring RHAMM splice variants that
lack exon 4 and exon 13 preserve the HA-binding domain
]. To determine whether HA that is synthesized by
HAS1 interacts with RHAMM, HeLa cells were
transiently co-transfected with plasmids that express HAS1
along with GFP-tagged full-length RHAMM or a
splicevariant of RHAMM (exon 4). The transfected cells were
synchronized for Mitosis or G1/S cell cycle stages using
thymidine block, which was verified by flow cytometry
(Additional file 3: Figure S3B). The cells were
synchronized in mitosis (when the nuclear membrane is dissolved)
because at this stage cytoplasmic HA may have a higher
chance of interaction with the mitotic machinery. Total
cellular HA was captured using biotinylated bovine
HA-binding-protein (bHABP, Sigma) and streptavidin
-conjugated magnetic beads from the indicated cell
lysates (Fig. 6a). Half of the captured material was treated
with hyaluronidase (HAase, an HA degrading enzyme)
to remove any HA or its bound proteins. Both the
HAase-treated and untreated samples were subjected to
immunoblotting for RHAMM and HAS1. De novo
synthesized HA by HAS1 is covalently attached to HAS1
during its elongation [
], therefore HAS1 and any
protein(s) bound to HA can be isolated using bHABP [
As shown in Fig. 6a, we observed a differential, but
specific, association of RHAMM and its splice variant with
cellular HA during mitosis and G1/S phase. Neither
RHAMM nor HAS1 were detected when the captured
material was treated with HAase, indicating both of
these proteins are associated through cellular HA. This
result suggests that de novo HA synthesized by HAS1
interacts with RHAMM during mitosis, as well as G1/S
phases of cell cycles, possibly at different ratios.
Cytoplasmic RHAMM also interacts with BRCA1 [
irrespective of the cell-cycle stage. BRCA1 is one of the
most researched breast and ovarian cancer susceptibility
gene that has been found to be associated with γ-tubulin
of centrosome [
] and thus may act as an essential
component of spindle formation. We wanted to explore
whether HA-interacting RHAMM also associates with the
BRCA1 protein in these cells. GFP-tagged RHAMM
fulllength and its splice variants (exon 4 and exon 13) were
transiently expressed in HeLa cells and cell lysates were
incubated with biotinylated-HA as bait, followed by
streptavidin-bead-mediated capture. We also expressed
GFP-ΔNeurocan in HeLa cells as control. Neurocan is an
HA-binding protein and the HA-binding domain of
Neurocan is fused with GFP in the GFP-ΔNeurocan
expression construct [
]. The captured material (HA-binding
proteins) was digested with HAase to confirm that the
captured HA-associated proteins are directly or indirectly
associated with biotinylated-HA. Immunoblot analysis
showed that the GFP-RHAMM proteins and
GFPΔNeurocan were bound by the ‘bait’ HA (Fig. 6b). The
same immunoblot was reprobed with anti-BRCA1
antibody to identify the association of endogenous BRCA1
with HA-captured proteins. BRCA1 was found to be
associated with the HA-RHAMM complex, but not with
Neurocan-HA complex, suggesting that BRCA1 may
interact directly with RHAMM as BRCA1 does not
possess an HA-binding domain. These results demonstrate
the molecular interactions of HAS1-synthesized
cytoplasmic HA with RHAMM and possibly with BRCA1, which
are essential for proper spindle as well centrosomal
Hyaluronic acid (HA) is composed of repeating
disaccharide units and is synthesized by three different
membranebound synthase enzymes (HAS1, HAS2 and HAS3). HA
is present ubiquitously in the extracellular matrix and
undergoes rapid turnover. HAS1 expression is typically
very low in healthy cells [
3, 9, 13, 62, 63
] and found to be
upregulated in specific inflammatory conditions like
]. Histology and in vitro overexpression
data suggest that HAS1 accumulates significantly more in
the cytoplasmic space rather than on the plasma
1, 2, 13, 65, 66
]. Enzymatically-active splice variants
of HAS1 exclusively accumulate in the cytoplasm and
have the ability to retain the full-length HAS1 in the
cytoplasm because HAS1 multimerizes for its proper
enzymatic function [
Our results demonstrate that an increase in cytoplasmic
HA through overexpression of HAS1 in different human
cell types increases the characteristic cancer phenotypes
of clonal variation, EMT, micronucleation and centrosome
abnormalities such as clustering and/or fragmentation.
Published reports characterizing HAS3 did not describe
any such cytoplasmic HA production by HAS3 and its
related effects [
]. HAS2 and HAS3 were found to
produce extracellular HA , and HAS2 appears to
contribute to cytoplasmic HA production when HAS2 is
overexpressed in pancreatic cancer cells [
HAS2 expression is correlated with EMT [
HAS1 nor HAS3 have been directly implicated in EMT.
To our knowledge, no reports are found correlating
neither HAS2 nor HAS3 with clonal, variation,
micronucleation or centrosome abnormalities. Thus, our results
demonstrate unique HAS1-specific phenotype (clonal
variations, micronucleation and centrosome
abnormalities), which are not evident upon HAS2 overexpression.
The results presented here are reproducible in non-cancer
cells (MCF10A) and as well as with different cancer cell
lines (cervical and colon) as model systems. Our data
support the hypothesis that HAS1-synthesized cytoplasmic
HA correlates with the interference in genomic stability
and normal mitosis.
Centrosome abnormalities and CIN manifest in cells
as aneuploidy and/or micronucleation and often leads to
decreased survival, however surviving sub-clones may
suggest the emergence of therapy-resistant clones or
tumor stem cells [
47, 70, 71
]. Centrosome abnormalities
and/or micronucleation are the most
commonlydetected markers in most human cancers (solid and
]. Cell cycle, mitosis, DNA repair,
proliferation and tissue apicobasal polarity depend on
precise centrosome divisions and localization . The
integral relation of centrosome abnormality, its spatial
association with the Golgi and overall role in CIN has
been explored by many groups, who have sought to find
the mechanistic and molecular interactions driving
cancer initiation and development (reviewed in [
Nevertheless, how CIN contributes to cancer is not well
understood, notwithstanding the fact that the presence
of micronuclei indicates the extent of CIN progression,
eventually increasing the probability of clonal
diversification leading to cellular transformation [
cancer cells that possess clustered/enlarged centrosomes
and micronuclei may undergo abnormal cell divisions,
risking further genetic instability that contributing to
EMT, cancer progression and cancer stem cell initiation
. Our findings demonstrate for the first time that
cytoplasmic HA homeostasis is required to maintain
precise centrosome functions and to prevent both EMT,
and micronucleation and their subsequent effects on
We found expression of CD44 in cytoplasm and also to
some extent on the plasma membrane in MCF10A cells
only when HAS1 was expressed (Fig. 1b). This may
indicate CD44-mediated induction of EMT (as in Fig. 2)
and may correlate with CD44 associated cancer stem cell
]. Therefore we do not rule out the possibility
of CD44-expression mediated EMT. The exact
mechanism(s) that correlate CD44 with the observed
abnormalities prompt merit for further investigation.
Both full-length and splice variants of HAS1 and
RHAMM are overexpressed in multiple myeloma and
bladder cancers, but absent in healthy cells [
18, 39, 74
RHAMM is a cytoplasmic protein that is unconventionally
exported to the cell surface during wound repair, where it
binds with HA and activates CD44 resulting in
stimulation of the Ras/Erk1,2 pathway, a cascade that functions
in cellular proliferation and that often dysregulated in
]. The centrosomal-targeting domain of RHAMM
overlaps with its HA-binding domains [
RHAMM is found in multiple compartments, but its
association with the centrosome and mitotic spindle are the
best characterized [
interactions are required for normal spindle formation and
for passage through the G2/M stage of the cell cycle [
RHAMM, Aurora kinases and Polo-like kinase-1 are
centrosome-associated proteins that have important roles
in cell cycle progression, checkpoint control and mitosis.
TPX2, a spindle assembly factor, mediates AuroraA kinase
(AURKA) localization to spindle microtubules and
activates it by autophosphorylation [
]. Recent studies also
showed that BRCA1, RHAMM, AURKA and TPX2
interactions are mechanistically important for microtubular
reorganization during mitotic spindle formation and
apicobasal polarization for tissue organization [
]: all these
interactions highlight the critical and precise functions of
centrosome. These fundamental aspects of centrosome
functions are compromised in most malignancies, and our
results suggest that this could be due to an imbalance in
Previous work on HA has mainly focused on its
ECMrelated and signal transduction roles. So far, no studies
have reported any correlation between cytoplasmic HA
imbalance, micronucleation and centrosome abnormalities
in any cell type. A previously unknown role for
cytoplasmic HA that HAS1-synthesized cytoplasmic HA induce
EMT, micronucleation, centrosome abnormalities, and
aneuploidy in different cell types is supported with these
results. Although growing evidence indicates
accumulation of cytoplasmic HA during mitosis [
], a role for
cytopalsmic HA in genetic stability and cellular
transformation has not yet been reported.
Using different cells and expression systems as models, we
demonstrated that HAS1 expression induced clonal
diversity, multi-nucleus formation, micronucleus generation,
centrosome abnormalities which are the most common
cancer-associated cellular anomalies found in histological
studies. Our results also indicate that intracellular HA
produced by HAS1 is associated with the
BRCA1RHAMM-microtubule complex, which implies a possible
mechanistic role in cancer initiation and progression.
Overall, our findings that HA, a primitive
glycosaminoglycan influences EMT, centrosome abnormalities, and
micronucleation noteworthy and will contribute to a
better understanding of cancer biology and may eventually
lead to clinical and therapeutic opportunities.
Additional file 1: Figure S1. Expression of HAS1. (A) Lung primary cells
were transiently transfected with pCDNA3-A2-HAS1 or empty vector
(pCDNA3-A2) and subjected to HA fluorescence staining (green) after
72 h. Nucleus was stained with DAPI. (B) HeLa cells were engineered and
selected for Tetracycline-on inducible HAS1 expression. Cells were grown
in tetracycline-free media in 8-well chamber slides for 16 h followed by
with or without doxycycline (Dox) treatment for 40 h, and then HA
fluorescence staining (white) and nuclear staining with DAPI (blue).
(C) DLD1 cells were transfected and selected for Tet-inducible HAS1
expression. The cells were grown in tetracycline-free media followed by
induced with doxycycline (Dox) treatment for 40 h. The cells were stained
for HA localization using bHABP (Green). DLD1-pTET cells served as
negative control. (PDF 150 kb)
Additional file 2: Figure S2. Effect of HAS1 expression on mitotic
index and cell growth. (A) Lower mitotic index was observed in HAS1
expressing MCF10A cells in comparison to LMA2-expressing of mock
transfected cells. MCF10A cells transfected with the indicated cDNA in
pCDNA3. The selected populations were seeded onto 8-chamber glass
slides, incubated overnight, and then fixed and DAPI-stained to count
mitotic/non-mitotic nuclei based on the chromatin / nucleus structure.
HE-HAS1: HAS1 in pCDNA3 with N-terminal hemagglutinin fusion-tag,
A2-HAS1: HAS1 in pCDNA3 with N-terminal A2 fusion-tag, LMA2:
unrelated protozoa gene in pCDNA3 with C-terminal A2 fusion tag and
Mock: transfection without any plasmid and not selected with any
antibiotic. (B) HAS1 expressing cells showed the slower growth after
induction with Dox. HeLa cells engineered and selected for
Tetracyclineon inducible HAS1 or GFP expressing plasmids. The cell populations were
subjected to growth analysis to test the effect of inducible expression of
genes (GFP and HAS1) on growth for 13-days with Dox at different
concentrations. The results are presented as fold increase of viable cells
compared to seeded cells at Day 0. The growth of all HAS1-expressing
cells was slower than the GFP-puromycin-vector controls, may be due
to background synthesis (leakiness) of intracellular-HA by HAS1 even at
0 μg/ml Dox induction. At higher concentrations of Dox (6 μg/ml) the
growth cease beyond 10th day for HAS1 but not for control GFP.
(PDF 12 kb)
Additional file 3: Figure S3. (A) Larger Golgi apparatus were observed
in the cells expressing HAS1 (lower panels) as compared to control pTET
cells (upper panels). The tetracycline-inducible DLD1 cells with HAS1 and
control (pTET) as described in Fig. 5B were stained for Golgi bodies
(GM130, green), centrosome (pericentrin, red) and nucleus (blue) in the
first panel, and HA (white) in the second panel and DIC image of the
structure of the cell in third panel. (B) Respective cell populations indicate
the synchronized cells at mitosis and G1/S phase of the cell cycle.
Transfected HeLa cells were synchronized with double thymidine blocks.
The cells were measured for their DNA contents using flow cytometry to
verify synchronization. The cells were harvested, fixed with cold ethanol
and stained with propidium iodide to measure the content of DNA in
cell-populations. (PDF 158 kb)
AURKA: AuroraA kinase; bHABP: Biotinylated-hyalurone-binding-protein;
CIN: Chromosomal instability; Dox: Doxycycline; ECM: Extracellular matrix;
EMT: Epithelial-to-Mesenchymal Transition; HA: Hyaluronic acid;
HAase: Hyaluronidase; HAS1: Hyaluronic Acid Synthases 1; HAS2: Hyaluronic
Acid Synthases 2; HAS3: Hyaluronic Acid Synthases 3;
IF: Immunofluorescence; RHAMM: Hyaluronan-mediated motility receptor;
ROI: Region of interest
Some of the plasmids used in this study were constructed while I was
working with Prof. Linda Pilarski (University of Alberta, Canada). Prof. Pilarski
also guides us with technical helps. We are grateful to Dr. Steve Lewis and
Dr. Michael Wall for their critical review of the manuscript. We also thank
New Brunswick Health Research foundation for finance.
New Brunswick Health Research foundation supported with the research
grants. There is no role of the funding body in the design of the study and
collection, analysis, and interpretation of data and in writing the manuscript.
Availability of data and materials
The datasets used and/or analysed during the current study are available
from the corresponding author on reasonable request.
NN, AK, and SC: Performed the experiment and analyzed the data. AG:
Conceived the idea, designed and performed the experiments, and analyzed
the data. NN, AK, RJO, and AG: Prepared the manuscript. All the authors read
and approved the manuscript.
Ethics approval and consent to participate
Consent for publication
The authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
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