Hakai reduces cell-substratum adhesion and increases epithelial cell invasion.

Abstract

The dynamic regulation of cell-cell adhesions is crucial for developmental processes, including tissue formation, differentiation and motility. Adherens junctions are important components of the junctional complex between cells and are necessary for maintaining cell homeostasis and normal tissue architecture. E-cadherin is the prototype and best-characterized protein member of adherens junctions in mammalian epithelial cells. Regarded as a tumour suppressor, E-cadherin loss is associated with poor prognosis in carcinoma. The E3 ubiquitin-ligase Hakai was the first reported posttranslational regulator of the E-cadherin complex. Hakai specifically targetted E-cadherin for internalization and degradation and thereby lowered epithelial cell-cell contact. Hakai was also implicated in controlling proliferation, and promoted cancer-related gene expression by increasing the binding of RNA-binding protein PSF to RNAs encoding oncogenic proteins. We sought to investigate the possible implication of Hakai in cell-substratum adhesions and invasion in epithelial cells.
Parental MDCK cells and MDCK cells stably overexpressing Hakai were used to analyse cell-substratum adhesion and invasion capabilities. Western blot and immunofluoresecence analyses were performed to assess the roles of Paxillin, FAK and Vinculin in cell-substratum adhesion. The role of the proteasome in controlling cell-substratum adhesion was studied using two proteasome inhibitors, lactacystin and MG132. To study the molecular mechanisms controlling Paxillin expression, MDCK cells expressing E-cadherin shRNA in a tetracycline-inducible manner was employed.
Here, we present evidence that implicate Hakai in reducing cell-substratum adhesion and increasing epithelial cell invasion, two hallmark features of cancer progression and metastasis. Paxillin, an important protein component of the cell-matrix adhesion, was completely absent from focal adhesions and focal contacts in Hakai-overexpressing MDCK cells. The expression of Paxillin was found to be regulated by a proteasome-independent mechanism, possibly due to the decreased abundance of E-cadherin.
Taken together, these results suggest that Hakai may be involved in two hallmark aspects of tumour progression, the lowering cell-substratum adhesion and the enhancement of cell invasion.

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RESEARCH ARTICLEOpen Access
Hakai reduces cell-substratum adhesion and
increases epithelial cell invasion
Teresa Rodríguez-Rigueiro1, Manuel Valladares-Ayerbes2, Mar Haz-Conde1, Luis A Aparicio2and
Angélica Figueroa1*
Abstract
Background: The dynamic regulation of cell-cell adhesions is crucial for developmental processes, including tissue
formation, differentiation and motility. Adherens junctions are important components of the junctional complex
between cells and are necessary for maintaining cell homeostasis and normal tissue architecture. E-cadherin is the
prototype and best-characterized protein member of adherens junctions in mammalian epithelial cells. Regarded as
a tumour suppressor, E-cadherin loss is associated with poor prognosis in carcinoma. The E3 ubiquitin-ligase Hakai
was the first reported posttranslational regulator of the E-cadherin complex. Hakai specifically targetted E-cadherin
for internalization and degradation and thereby lowered epithelial cell-cell contact. Hakai was also implicated in
controlling proliferation, and promoted cancer-related gene expression by increasing the binding of RNA-binding
protein PSF to RNAs encoding oncogenic proteins. We sought to investigate the possible implication of Hakai in
cell-substratum adhesions and invasion in epithelial cells.
Methods: Parental MDCK cells and MDCK cells stably overexpressing Hakai were used to analyse cell-substratum
adhesion and invasion capabilities. Western blot and immunofluoresecence analyses were performed to assess the
roles of Paxillin, FAK and Vinculin in cell-substratum adhesion. The role of the proteasome in controlling cell-
substratum adhesion was studied using two proteasome inhibitors, lactacystin and MG132. To study the molecular
mechanisms controlling Paxillin expression, MDCK cells expressing E-cadherin shRNA in a tetracycline-inducible
manner was employed.
Results: Here, we present evidence that implicate Hakai in reducing cell-substratum adhesion and increasing
epithelial cell invasion, two hallmark features of cancer progression and metastasis. Paxillin, an important protein
component of the cell-matrix adhesion, was completely absent from focal adhesions and focal contacts in Hakai-
overexpressing MDCK cells. The expression of Paxillin was found to be regulated by a proteasome-independent
mechanism, possibly due to the decreased abundance of E-cadherin.
Conclusions: Taken together, these results suggest that Hakai may be involved in two hallmark aspects of tumour
progression, the lowering cell-substratum adhesion and the enhancement of cell invasion.
Background
The most frequent types of tumours are carcinomas,
which develop through the transformation of epithelial
cells. Epithelial cells are polarized and are often con-
nected with each other via cell-cell adhesions to form
structured cells sheets. The formation of these tight and
compact cell-cell adhesions restricts cell movement
within the epithelium. Epithelial cells are also attached
to an extracellular matrix substratum which is essential
for their differentiation and polarization [1]. During
transformation, epithelial cells start to proliferate,
acquire the ability to migrate, and lose both the intercel-
lular adhesion, mediated by cadherins at adherens junc-
tions, and the interactions with the extracellular matrix.
All of these characteristics facilitate the invasion and
metastasis of epithelial cells [2].
Hakai was firstly described as a RING finger-type E3
ubiquitin-ligase for the E-cadherin complex [3]. E-cad-
herin is the prototypical and best characterized member
* Correspondence: angelica.figueroa.conde-valvis@sergas.es
1Translational Cancer Research Group, Instituto de Investigación Biomédica A
Coruña (INIBIC), Complejo Hospitalario Universitario A Coruña (CHUAC),
SERGAS, A Coruña, Spain
Full list of author information is available at the end of the article
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© 2011 Rodríguez-Rigueiro et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited.

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of the cadherins at adherens junctions and its loss is in
carcinoma associated with poor prognosis [4]. Hakai
binds to the cytoplasmic domain of E-cadherin and
mediates its ubiquitination, endocytosis and degradation
[3,5]. In consequence, it leads to the disruption of
epithelial cell-cell adhesions, inducing epithelial-
mesenchymal transition (EMT), a key event in epithelial
transformation [6,7]. Apart from this functional role, we
recently reported that Hakai is not only implicated in
lowering cell-cell contacts, but can also promote prolif-
eration in an E-cadherin-independent manner. More-
over,Hakaioverexpression
independent cell growth, produces protrusions that are
dynamically extended and retracted, and increases the
ability of the RNA-binding protein PSF to bind to
mRNAs that encode cancer-related proteins [8,9].
Given the role of Hakai in tumorigenesis, we are inter-
ested to examine the possible implication of Hakai in
the regulation of adhesions to the extracellular matrix
(ECM) and invasion in epithelial cells, two hallmark
processes in cancer and metastasis [10,11]. To investi-
gate this possibility, we studied Hakai’s influence on cell
attachment to the substrate and invasion capacity of
Madin-Darby canine kidney (MDCK) cells. We report
that in this system, Hakai overexpression leads to a
reduction in cell adhesion to the substrate with impact
on the key focal adhesion-associated protein Paxillin,
and increases cell invasion. Our data suggest that Hakai
may have an important functional role during oncogen-
esis through its implication on cell adhesion to the sub-
strate and cell invasion.
inducesanchorage-
Methods
Antibodies and materials
The rabbit polyclonal anti-Hakai antibody (Hakai-2498)
was kindly provided by Yasuyuki Fujita [8]. Anti-FAK
antibody was from Upstate (Charlottesville, VA), anti-
Paxillin antibody was from Transduction Laboratories,
anti-Vinculin was from Chemicon International (Hamp-
shire, UK), antibody to the extracellular portion of E-
cadherin (ECCD-2) was from Zymed (South San Fran-
cisco, CA), anti-a-Tubulin antibody was from Immuno-
logicals Direct (Oxford Biotechnology Ltd., UK), HRP-
rabbit and mouse polyclonal antibodies was from GE
Healthcare (UK) and Alexa-Fluor 488 mouse antibody
and Alexa-mouse 568 were from Molecular Probes
(Paisley, UK). All primary antibodies were used at dilu-
tions of 1:1000 for Western blotting and 1:100 for
immunofluorescence, except for anti-FAK antibody that
was used at a dilution of 1:5000 for Western blotting
and 1:500 for immunofluorescence. Secondary antibo-
dies were used at dilutions 1:10000 for HRP-rabbit,
1:5000 for HRP-mouse antibody, and 1:200 for Alexa-
Fluor antibodies. pcDNA-HA-Hakai was kindly provided
by Walter Birchmeier [3]. MG132 and lactacystin were
obtained from Calbiochem (Darmstadt, Germany).
Cell culture
MDCK cells were cultured in Dulbecco’s modified
Eagle’s medium (DMEM) containing penicillin/strepto-
mycin and 10% FCS at 37°C and ambient air supple-
mented with 5% CO2. MDCK cells stably expressing
Hakai were kindly provided by Yasuyuki Fujita [8].
MDCK cells were transfected with pcDNA-HA-Hakai
using Lipofectamine-Plus™ reagent (Invitrogen) and
were selected in a medium containing 800 μg ml-1of
G418 (Calbiochem). More than ten stable clones were
obtained from two independent transfections. All clones,
including 4, 6, 7 and 8 clones, represented comparable
phenotypes and the results shown were obtained using
clone 4, unless otherwise indicated. MDCK cells stably
expressing E-cadherin shRNA in a tetracycline-inducible
manner were kindly provided by Yasuyuki Fujita [12].
Adhesion assay
For the adhesion assay, 1 × 105cells were plated in a 6-
well plate using human fibronectin-coated coverslips
(BD Bioscience, Belgium). After 24 hours, the cells were
vigorously washed five times with phosphate-buffered
saline (PBS), and the remaining cells attached to the
plate were analyzed by phase contrast images using a
Leica DMIRB microscope with a Hamamatsu C4742-95
Orca camera.
Western blotting
Cells cultured in 6-well plate dishes were lysed for 30
min in 0.3 ml of 1% Triton X-100 lysis buffer (20 mM
Tris-HCL [pH 7.5], 150 mM NaCl, and 1% Triton X-
100) containing 5 μg ml-1lupeptin, 50 mM phenyl-
methylsulfonyl fluoride, and 7.2 trypsin inhibitor units
for aprotinin. After centrifugation at 14, 000 × g for 10
min, thirty micrograms of the supernatants were loaded
in 10% polyacrilamide SDS-PAGE. Western blotting was
performed as previously described [13].
Immunofluorescence
For immunofluorescence, human fibronectin-coated cov-
erslips (BD Bioscience, Belgium) were used. Cells on cov-
erslips were washed twice in PBS and incubated in 3%
parafolmaldehyde-PBS for 15 min. After washing twice in
PBS, cells were permeabilized in 0.5% Triton X-100-PBS
for 15 min, followed by blocking in DMEM containing
10% fetal bovine serum for 1 h. They were further incu-
bated in primary antibodies for 1 h, washed in PBS, incu-
bated in Alexa-Fluor 488 or Alexa-Fluor 568-conjugated
secondary antibody solution for 1 h, and washed four
times in PBS. The coverslip was the mounted onto
Mowiol on a glass slide. Immunofluorescent images were
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analyzed by epifluorescence microscopy. To obtain epi-
fluorescence images, we used a Zeiss Axioskop 1 with a
Roper Scientific Coolsnap camera.
Invasion assay
For invasion assays, 5 × 104cells were plated in DMEM/
1% FCS in a cell invasion chamber (Cell invasion assay
kit, Chemicon International) in a 24-well plate, which
contains an 8-μm pore size polycarbonate membrane
covered with a thin layer of collagen matrix. Invasive
cells migrate through a membrane according to the gra-
dient of FCS to the lower chamber that contains
DMEM/10% FCS. The invaded cells were stained with
crystal violet, and the absorbance of stained cells was
measured in a 565-nm wavelength.
Statistical analysis
Student t test assuming unequal variance was performed
for statistical analysis.
Results and Discussion
Hakai overexpression reduces cell-substratum adhesion
In a recent study, we reported that MDCK cell lines
overexpressing Hakai (4-6-fold higher than endogenous
Hakai) exhibited reduced cell-cell contacts and higher
cell proliferation than non-transfected parental MDCK
cells [8]. Here, we examined the effect of Hakai overex-
pression on cell-substratum adhesions. When we incu-
bated several clones of Hakai-overexpressing cells, we
realized that they had reduced adhesions to the substra-
tum. Parental MDCK cells attached tightly to the sub-
stratum and washes with PSB did not detach the cells
from the plate (Figure 1, upper panel). In these cells,
trypsin was required to detach them from the plate. In
contrast, Hakai-overexpressing MDCK cells were easily
detached from the plate by washing with PBS (Figure 1,
lower panel). This observation suggests that overexpres-
sion of Hakai leads to a reduction of cell-substratum
adhesions.
Then, we examined key protein components of cell
adhesion, such as FAK, Paxillin and Vinculin, which
localize to focal adhesions and focal contacts, mediate
adhesion to the substratum, and participate in initiating
signal transduction pathways [14]. By Western blotting,
we found that Paxillin expression was strongly reduced
in Hakai-overexpressing cells, whereas the levels of FAK
and Vinculin were not affected by Hakai expression
(Figure 2A). Further analysis of the adhesions by immu-
nofluorescence revealed that FAK protein localized at
focal adhesions and focal contacts in parental MDCK
cells, while in Hakai-overexpressing cells, FAK was dif-
fusely distributed in the cytoplasm, enriched at the sites
of focal contacts, and absent from focal adhesions (Fig-
ure 2B). As expected, in Hakai-overexpressing cells,
Paxillin was undetectable (Figure 2B). These data sug-
gest that cell-substratum adhesions and the formation of
focal adhesions are highly dependent on overexpression
of Hakai, probably due to the specific downregulation of
Paxillin. Although Paxillin primarily functions as a
molecular adapter or scaffold protein that provides mul-
tiple docking sites at the plasma membrane, it is also
important for the transduction of external signals to eli-
cit changes in cell motility and modulate gene expres-
sion by the various MAP kinase cascades. Paxillin binds
to many proteins that are involved in effecting changes
in the organization of the actin cytoskeleton, and are
thus necessary for cell motility associated with tumor
metastasis [15]. Therefore, the complete absence of Pax-
illin at focal adhesions may be one of the mechanisms
that explain the loss of cell adhesion to the substrate.
Paxillin down-regulation is controlled by a proteasome-
independent mechanism and results from the decreased
expression of E-cadherin
To investigate whether the reduced Paxillin levels found
in Hakai-overexpressing cells was the result of increased
degradation of Paxillin protein, we analyzed the effect of
proteasome inhibitors MG132 and lactacystin in Hakai-
overexpressing and parental MDCK cells. None of these
inhibitors affected Paxillin abundance in Hakai-overex-
pressing cells (Figure 3), indicating that the expression
of Paxillin is controlled in a proteasome-independent
manner. We therefore ask whether the loss of E-
Figure 1 Effect of Hakai overexpression on the adhesion to the
substrate in parental and Hakai-overexpressing MDCK cells.
Cells were plated on human fibronectin-coated coverslips and after
24 h of plating they were vigorously washed 5 times with PBS, and
examined by phase contrast. Scale bars, 10 μm.
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cadherin was sufficient to reduce Paxillin expression
levels. Using MDCK cells in which E-cadhering was
knocked down by shRNA in a tetracycline-regulated
manner, Paxilllin expression was strongly reduced where
E-cadherin was silenced (Figure 4). These findings sug-
gest that the loss of Paxillin in Hakai-overexpressing
cells may, at least partially, result from the decreased
expression level of E-cadherin.
However, several lines of evidence suggest that Hakai
can also affect cellular phenotypes in an E-cadherin-
independent manner. First, MDCK cells expressing E-
cadherin shRNA do not extend spiky protrusions that
are seen in Hakai-overexpressing cells [12]. Second, in
Hakai-overexpressing MDCK cells, Hakai is localized at
the end of the protrusion where FAK is also enriched
[8], suggesting that Hakai may be involved in regulating
the dynamic extension and retraction of these structures
and in influencing cell motility and cell attachment to
the ECM. Finally, Hakai is also located in the nucleus,
where it can interact with nuclear proteins such as PSF
and modulate cancer-related gene expression [8] and it
may act as a correpresor of the estrogen receptor in
breast cancer cells [16]. These findings suggest that
Hakai can have ubiquitin-independent functions that
Figure 3 Effect of proteasome inhibitors on Paxillin expression.
Parental or Hakai-overexpressing MDCK cells were incubated for 6 h
in the absence or presence of proteasome inhibitors (20 μM MG132
or 5 μM lactacystin), and cell lysates were examined by Western
blotting to detect Paxillin.
Figure 4 Effect of E-cadherin knockdown on Paxillin
expression. MDCK pTR cells stably expressing E-cadherin shRNA
were incubated for 3 days in the presence of tetracycline, and were
analyzed by immunofluorescence to detect E-cadherin and Paxillin.
The expression of E-cadherin shRNA is induced only in 30-40% of
total cells in this cell line, and the knockdown of E-cadherin is
observed in the right half of the presented image. Data are
representative of three independent experiments. Scale bar, 20 μm.
B
MDCK MDCK
HA-Hakai
FAK
Paxillin
A
α−Tubulin
FAK
Paxillin
Vinculin
7
MDCK
468
MDCK HA-Hakai
Figure 2 Effect of overexpression of Hakai on the levels and localization of FAK, Paxillin and Vinculin. (A) Expression of FAK, Paxillin and
Vinculin in parental and in several Hakai-overexpressing MDCK checked clones (described in methods). Lysates prepared from the indicated cells
were examined by Western blot analysis to detect the proteins shown. (B) Effect of overexpression of Hakai on the localization of FAK and
Paxillin. Parental and Hakai-overexpressing MDCK cells were examined by immunofluorescence with anti-FAK and anti-Paxillin antibodies. Scale
bars, 10 μm.
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may influence the cell phenotype, in addition to its
influence on known substrates (like E-cadherin) and as-
yet unreported substrates in the nucleus or the cyto-
plasm, as previously reported for other E3-ubiquitin
ligases [17,18].
Hakai overexpression enhances invasiveness
In closing, we examined whether the overexpression of
Hakai also affect the invasiveness of cells. We used a
polycarbonate membrane over which a thin layer of
extracellular matrix (ECM) was applied to serve as an in
vitro basement membrane. Since only invasive cells are
capable of migrating through the ECM layer, we ana-
lyzed the ability of parental and Hakai-overexpressing
MDCK cells to invade this in vitro matrix. Staining of
the cells that passed through the membrane revealed
that, whereas parental cells did not invade the matrix,
Hakai-overexpressing cells readily passed through the
membrane (Figure 5), indicating that overexpression of
Hakai also enhances cell invasion. Taken all together,
these observations suggest that Hakai may be involved
in the regulation of invasion and cell-substratum adhe-
sion. However, further investigations of Hakai in in vitro
and in vivo model systems would lead us to validate its
role during tumorogensis. Therefore, Hakai is implicated
in several processes that are often occurring during
epithelial-mesenchymal transition (EMT). In the last few
years, it has been proposed that EMT mRNAs markers
can be detected in circulating tumour cells (CTC) in the
blood from cancer patients, helping to select new thera-
peutic targets with important clinical implications [19]
Indeed, our group has recently proposed Plakophillin-3
mRNA, involved in epithelial cell-cell adhesions, as a
biomarker for detection of CTCs in gastrointestinal can-
cer patients [20]. Further investigation is warranted to
elucidate the molecular mechanism whereby Hakai
influences these processes under physiological and
pathological conditions, and to assess the potential use-
fulness of Hakai as therapeutic target for cancer.
Conclusions
In summary, we present evidences that indicate that
Hakai is implicated in the regulation of cell-substratum
adhesions and in epithelial cell invasion. We have
shown that the overexpression of Hakai in epithelial
cells reduces Paxillin expression level and it disappears
from focal adhesions and focal contacts. We demon-
strated that this Paxillin reduction can not be recovered
by using two important proteasome inhibitors, such as
lactacystin and MG132, further supporting that Paxillin
down-regulation is controlled by a proteasome-indepen-
dent mechanism. Furthermore, we have shown that pax-
illin reduction may, at least partially, result from
decreased expression level of E-cadherin. All these data
suggest the implication of Hakai in controlling several
important cellular processes that are crucial during
tumour progression.
Acknowledgements
We thank Myriam Gorospe for critical reading the manuscript. We thank
Yasuyuki Fujita for helpful discussions and for providing reagents. T.R.R and
A.F. are recipients of a grant from the Secretaria Xeral I+D+I, Xunta de
Galicia, Spain (10CSA916023PR and IPP.08-07) and M.H.C is supported by the
Instituto de Salud Carlos III, Spain (CA09/00116). This work is supported by a
grant from the Conselleria de Sanidade (PS09/24) and from the Secretaria
Xeral I+D+I, (10CSA916023PR), both from Xunta de Galicia, Spain.
Author details
1Translational Cancer Research Group, Instituto de Investigación Biomédica A
Coruña (INIBIC), Complejo Hospitalario Universitario A Coruña (CHUAC),
SERGAS, A Coruña, Spain.2Servizo Oncología Médica, Complejo Hospitalario
Universitario A Coruña (CHUAC), SERGAS, A Coruña, Spain.
Authors’ contributions
AF, TRR and MCC contributed to experimental research. AF contributed to
the design, the analysis of the results and writing the manuscript. All the
authors have given the discussion, critical reading of the manuscript and
final approval of the version to be published.
Competing interests
The authors declare that they have no competing interests.
Received: 12 August 2011 Accepted: 3 November 2011
Published: 3 November 2011
References
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Figure 5 Effect of Hakai overexpression on cell invasion.
Parental and Hakai-overexpressing MDCK cells were plated in an
invasion chamber. After 24 h of incubation, invaded cells were
stained with crystal violet and the absorbance of the stained cells
was measured. Lower panels represent the staining of the migrating
cells through the extracellular matrix. Data are represented as the
means ± SD.
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