Alpha-enolase (ENO1) controls alpha v/beta 3 integrin expression and regulates pancreatic cancer adhesion, invasion, and metastasis
Principe et al. Journal of Hematology & Oncology
Alpha-enolase (ENO1) controls alpha v/beta 3 integrin expression and regulates pancreatic cancer adhesion, invasion, and metastasis
Moitza Principe 0 1 3
Simone Borgoni 0 1 3
Mariafrancesca Cascione 2 7
Michelle Samuel Chattaragada 0 1 3
Sammy Ferri-Borgogno 0 1 3
Michela Capello 0 1 3
Sara Bulfamante 0 1 3
Jennifer Chapelle 1 6
Francesca Di Modugno 5
Paola Defilippi 1 6
Paola Nisticò 5
Paola Cappello 0 1 3 6
Chiara Riganti 4
Stefano Leporatti 8
Francesco Novelli 0 1 3 6
0 Center for Experimental Research and Medical Studies (CeRMS), Azienda Universitaria Ospedaliera Città della Salute e della Scienza di Torino , Via Santena 5, 10126 Turin , Italy
1 Department of Molecular Biotechnology and Health Sciences, University of Turin , Turin , Italy
2 Dipartimento di Matematica e Fisica “Ennio De Giorgi”, Università del Salento , Lecce , Italy
3 Center for Experimental Research and Medical Studies (CeRMS), Azienda Universitaria Ospedaliera Città della Salute e della Scienza di Torino , Via Santena 5, 10126 Turin , Italy
4 Department of Oncology, University of Turin , Turin , Italy
5 Regina Elena National Cancer Institute , Rome , Italy
6 Molecular Biotechnology Center, University of Turin , Turin , Italy
7 Euromediterranean Center for Nanomaterial Modelling and Technology (ECMT) of the Consiglio Nazionale delle Ricerche, Istituto Nanoscienze , Lecce , Italy
8 CNR Nantotec-Istituto di Nanotecnologia, Polo di Nanotecnologia c/o Campus Ecoteckne , Lecce , Italy
Background: We have previously shown that in pancreatic ductal adenocarcinoma (PDA) cells, the glycolytic enzyme alpha-enolase (ENO1) also acts as a plasminogen receptor and promotes invasion and metastasis formation. Moreover, ENO1 silencing in PDA cells induces oxidative stress, senescence and profoundly modifies PDA cell metabolism. Although anti-ENO1 antibody inhibits PDA cell migration and invasion, little is known about the role of ENO1 in regulating cell-cell and cell-matrix contacts. We therefore investigated the effect of ENO1 silencing on the modulation of cell morphology, adhesion to matrix substrates, cell invasiveness, and metastatic ability. Methods: The membrane and cytoskeleton modifications that occurred in ENO1-silenced (shENO1) PDA cells were investigated by a combination of confocal microscopy and atomic force microscopy (AFM). The effect of ENO1 silencing was then evaluated by phenotypic and functional experiments to identify the role of ENO1 in adhesion, migration, and invasion, as well as in senescence and apoptosis. The experimental results were then validated in a mouse model. Results: We observed a significant increase in the roughness of the cell membrane due to ENO1 silencing, a feature associated with an impaired ability to migrate and invade, along with a significant downregulation of proteins involved in cell-cell and cell-matrix adhesion, including alpha v/beta 3 integrin in shENO1 PDA cells. These changes impaired the ability of shENO1 cells to adhere to Collagen I and IV and Fibronectin and caused an increase in RGD-independent adhesion to vitronectin (VN) via urokinase plasminogen activator receptor (uPAR). Binding of uPAR to VN triggers integrin-mediated signals, which result in ERK1-2 and RAC activation, accumulation of ROS, and senescence. In shENO1 cancer cells, the use of an anti-uPAR antibody caused significant reduction of ROS production and senescence. Overall, a decrease of in vitro and in vivo cell migration and invasion of shENO1 PDA cells was observed. Conclusion: These data demonstrate that ENO1 promotes PDA survival, migration, and metastasis through cooperation with integrins and uPAR.
Pancreatic cancer; ENO1; Integrin; Atomic force microscopy; Invasion
Pancreatic ductal adenocarcinoma (PDA) is one of the
most lethal forms of cancer with a 5-year survival rate of
less than 8% . No early detection tests are currently
available and, as a result, most patients (80–85%) are not
diagnosed until late-stages of the disease, when the
cancer has metastasized to other organs [2–4]. In recent
years, many proteins have been proposed as new
immunological and molecular targets for PDA [5–7] and
among these, one of the most promising is alpha-enolase
In PDA and other tumors, ENO1 has a multifunctional
role depending on its localization [8, 9]. In addition to its
well-known enzymatic function during glycolysis, ENO1
acts as a plasminogen receptor on the cell surface [8, 10]
promoting metastatic cancer invasion [11–15]. We have
previously demonstrated that the injection of adenovirus
expressing cDNA coding for monoclonal antibody that
block the binding of ENO1 with plasminogen-inhibited
metastases formation of PDA cells in vivo . Many
metabolic enzymes, including ENO1, are known to interact with
cytoskeletal proteins (e.g., F-actin, tubulin), and these
associations provide ATP to promote the migration of tumor
cells [16, 17]. Although the roles of ENO1 as a glycolytic
enzyme [18, 19] and as a plasminogen receptor [12, 20]
have been well characterized, its role in the regulation of
cytoskeleton reorganization, particularly in PDA cells, has
not been fully clarified. Integrins regulate
adhesiondependent growth and invasion of tumor cells and the
integrin alpha v/beta 3 has been reported as a mediator of
anchorage independence , and its expression has been
associated with an aggressive form of disease in different
human tumors including PDA [22–27]. In this study, we
employed biochemical and functional approaches to
investigate molecules involved in cell adhesion and migration of
ENO1-silenced (shENO1) PDA cells. AFM (atomic force
microscopy) was employed to investigate the
nanostructural properties of shENO1 PDA cells. In addition, as cell
adhesion, survival and migration are dependent on integrin
binding to the extracellular matrix (ECM), and subsequent
signals, the roles of alpha V/beta 3 and alpha 5/beta
1 integrins, as well as uPAR (an ECM receptor) were
evaluated in shENO1 PDA cells. Moreover, the impact
of ENO1 silencing in the in vitro and in vivo invasion
and spreading of PDA cells was evaluated. Results
from this study indicated that ENO1, by cooperating
with integrins and uPAR, is a key regulator of cell
survival, adhesion, and motility in PDA.
10% FBS (Lonza), L-Glutamine (GE Healthcare, Milan,
Italy) and 50 μg/ml of gentamicin (Gentalyn 40 mg/ml,
Essex Italia, Segrate, MI, Italy) at 37 °C in a 5% CO2
atmosphere. Cells were detached using a 1 mM EDTA
solution in phosphate-buffered saline (PBS).
Silencing of ENO1 in PDA cell lines
ENO1 was silenced in PDA cell lines using a short
hairpin RNA (shRNA), as previously described .
Total RNA was extracted using the RNeasy Mini kit
(Qiagen, Milan, Italy) and reverse transcription was
performed from 1 μg of total RNA using the iScript cDNA
synthesis kit (BioRad, Segrate, MI, Italy), according to
the manufacturer’s instructions. Quantitative RT-PCR
was performed using SYBR Green dye (Life
Technologies, Monza, Italy) on a Thermal iCycler (BioRad). PCR
reactions were performed in triplicate, and the relative
amount of cDNA was calculated by the comparative
CT method using β-actin RNA sequences as a
control. Data are represented as mean ± SEM (standard
error of the mean) of three independent experiments.
Oligonucleotide primer sequences for Sybr Green
qRT-PCR are in the Supplementary Materials and
Methods (Additional file 1: Table S1).
Images of PDA cells were acquired by using CAT
(confocal-atomic force-total internal reflection fluorescence)
microscopy, which is a combination of an advanced
scanning probe microscope (Bioscope Catalyst, Bruker
Inc. USA), a confocal microscope (LSM 700, Zeiss
Germany), and a total internal reflection fluorescence
microscope (Laser TIRF 3, Zeiss). These devices were
mounted on an inverted microscope (Zeiss ObserverZ1,
Zeiss). In this study, CAT was used to evaluate
topography on living cell surfaces through AFM and
internal organization of fibers into cytoskeleton by
For topography acquisition, cells were cultured in plastic
Petri dishes (Corning by Sigma-Aldrich), as previously
described, and were left to grow until 70–80% confluent.
Immediately before performing measurements, cells
were washed with PBS solution and the medium was
replaced with 5 ml of Lebovitz culture medium (L15,
Sigma-Aldrich). Topographic and deflection images were
acquired on living cells for up to 2 h in contact mode, as
previously described . By using V-shaped silicon
nitride cantilevers (MSNL-10 VEECO, USA), probes were
chosen on the basis of a low range of elastic constant
value (nominal constants from 0.01 to 0.03 N*nm−1).
Topographic images were acquired at high resolution
(512*512 points*line) on a 50*50 μm2 area, and were
used to calculate the roughness values using the
Nanoscope Analysis Software. Prior to calculating the
roughness, all images were firstly treated with a second order
planefit, then with a second order flattening for deleting
every bow and minimizing sample three-dimensionality
effects. The roughness was evaluated on 25 areas of
1 μm*1 μm, separately acquired on nuclear regions and
cytoplasmatic regions. The mean value and its standard
deviation were obtained in this way.
For the confocal microscopy study, shCTRL and shENO1
cells were seeded at 4 × 104 cells/chamber well and were
incubated at 37 °C in a humidified 5% CO2 atmosphere. After
24 h, the medium was removed, and cells were washed
three times with PBS. Finally, cells were fixed with 4%
paraformaldehyde in PBS for 20 min at room temperature and
permeabilized with 0.1%Triton X-100 in PBS for 5 min at
room temperature. Cells were incubated with anti-ENO1
mAb at a dilution of 1:1000, followed by an Alexa Fluor
488-conjugated goat anti-mouse IgG (H + L) (Life
Technologies) at a dilution of 1:250, each for 1 h at room
temperature. For actin staining,
Phalloidin–Tetramethylrhodamine B isothiocyanate (TRITC) (Sigma-Aldrich) was
used at a dilution of 1:1000 for 30 min at room
temperature. Cells were then washed with PBS, and
samples were covered with a glass slide using Fluoroshiel with
DAPI (Sigma-Aldrich) as a mounting medium. DAPI was
used for nucleus staining. Samples were kept at 4 °C in the
dark until microscopic examination. Confocal acquisitions
were performed by using a 100×, 1.46 numerical aperture
oil immersion objective. Laser beams with 405, 488, and
555 nm excitation wavelengths were used to detect the blue
fluorescence from DAPI, green fluorescence from
AlexaFluo 488, and red fluorescence from TRITC,
respectively. Finally, confocal data files were processed
using ZEN software (Zeiss).
For cell attachment to matrix assays, Vitronectin,
Fibronectin, Collagen I, and IV (all from Sigma-Aldrich) were
diluted to 10 μg/ml in PBS and adsorbed onto 96-well
dishes at 4 °C overnight and then blocked for 2 h at 37 °
C with 2% BSA in PBS. Wells were then washed three
times with PBS before adding cells. PDA cells, either
shCTRL or shENO1, were harvested, centrifuged briefly,
then resuspended at a density of 5 × 104 cells/ml in
DMEM with 2% FBS. Cells were then seeded onto the
coated wells and allowed to adhere for 1 h at 37 °C in
the presence or absence of anti-human CD61 (beta3
integrin-Santa Cruz Biotechnology) at a concentration of
10 μg/ml. Non-adherent cells were removed by rinsing
with PBS three times. Cells were then fixed with 2%
glutaraldehyde (Sigma-Aldrich) in PBS for 20 min and
stained with crystal violet (Sigma-Aldrich). Stained cells
were washed with PBS, and the dye was solubilized with
10% acetic acid (Sigma-Aldrich) for 5 min on a rocker.
Attachment was quantified by measuring the absorbance
at 570 nm.
Western blot analyses
PDA cells were harvested, lysed, resolved, and
transferred to nitrocellulose membranes, as previously
described . Membranes were incubated overnight at
4 °C with the following antibodies (all diluted in TTBS
with 5% BSA): mouse anti-human integrin alpha v
(1:500, clone L230 made in-house), mouse anti-human
FAK (1:1000, made in-house), mouse anti-human
integrin alpha 5 and rabbit anti-human Src (both at 1:1000,
Santa Cruz Biotechnology by D.B.A. Italia, Segrate, MI,
Italy), mouse anti-human integrin beta 1 (1:500, BD
Bioscience, Buccinasco, MI, Italy), mouse anti-human
RAC1 (1:1000 Millipore by D.B.A Segrate, MI, Italy),
rabbit anti-human ERK1-2 (1:500, GeneTex by Prodotti
Gianni, Milan, Italy); rabbit anti-human integrin beta 3,
rabbit anti-human uPAR, rabbit anti-human phospho
ERK1-2, rabbit anti-human Paxillin, rabbit anti-human
phospho Paxillin, rabbit anti-human phospho FAK, rabbit
anti-human p38MAPK, rabbit anti-human phospho
p38MAPK and rabbit anti-human phospho Src (all
1:1000, Cell Signaling Technology by EuroClone, Pero,
MI, Italy).The mouse anti-human ENO1 (clone 72/1 )
and rabbit anti-human beta-actin antibody (Sigma
Aldrich, Milan, Italy), both at a dilution of 1:2000 in TTBS,
were incubated for 1 h at room temperature. Membranes
were then washed with TTBS and probed for 1 h at room
temperature with an HRP-conjugated anti-mouse IgG
(Santa Cruz Biotechnology) or an HRP-conjugated goat
anti-rabbit IgG secondary antibody (Sigma Aldrich),
accordingly, at a dilution of 1:2000. Immunodetection was
carried out by enhanced chemiluminescence using ECL
PLUS (GE Healthcare).
Flow cytometric analysis
A total of 1 × 105 cells were incubated with primary
antibody: mouse IgG1 anti-human CD51/61 (alpha v/
beta 3 integrin, BD, Milan, Italy), mouse IgG2a
antihuman CD41/61 (alpha IIb/beta 3 integrin, BioLegend
by Campoverde, Milan, Italy), mouse IgG1 anti-human
beta1 integrin (Santa Cruz Biotechnology) or an
isotype-matched negative control IgG1 or IgG2a
antibody (Ab) accordingly (Dako, Milan, Italy), all at doses
of 10 μg/ml for 30 min at 4 °C. After incubation with
primary Abs, cells were then incubated with a secondary
APC goat anti-mouse IgG Ab (BioLegend) for 20 min at
4 °C. Following this, cells were resuspended in PBS,
acquired with a BD Accuri C6 Flow Cytometer (BD) and
analyzed using FlowJo 7.5 software.
RAC GTPase activation assay
To perform the RAC assay, cells were washed twice on
ice with PBS and then lysed in 50 mM Tris, 150 mM
NaCl, 1% NP40, 10% glycerol, 10 Mm MgCl2, and
10 μg/ml each of leupeptin, pepstatin, and aprotinin.
Equal amounts of cell extracts were incubated at 4 °C
for 1 h with glutathione-coupled Sepharose 4B beads
(GE Healthcare) bound to recombinant GST-PAK CRIB
domain. Bound proteins were eluted in 2×
Laemmlireducing sample buffer and immunoblotted for RAC1.
Analysis of SA-β-galactosidase activity
For the senescence assay on extracellular matrices,
vitronectin, and collagen I (from Sigma-Aldrich) were coated
onto 96-well dishes, as described above. 3 × 104 cells/
well PDA cells, plus either shCTRL or shENO1, were
plated onto coated wells for 72 h in presence or absence
of 10 μg/ml anti-uPAR antibody (clone 3C6,
SigmaAldrich). Images were taken at microscope with ×10
objective. The SA-β-galactosidase activity was analyzed
as previously described .
PDA cells were cultured for 48 h in presence or absence
of 10 μg/ml anti-uPAR antibody (clone 3C6,
SigmaAldrich), and apoptotic cells were analyzed using
Annexin V Apoptosis Detection Kits (eBioscience by
Prodotti Gianni, Milan, Italy).
Scratch wound healing assay
PDA cells (1×105), plus either shCTRL or shENO1, were
seeded into each well of ibidi Culture-Inserts (Ibidi by
Giemme, Milan, Italy) and grown to confluence. After
12 h, a cell-free gap of about 500 μm was created after
removing the Culture-Insert, and cells were washed
twice with serum-free medium to remove any floating
cells. Cells which had migrated into the wounded area
or protruded from the border of the wound were
visualized and photographed under an inverted microscope at
each time point over a period of 24 h.
In vitro chemo-invasion assay
The invasive potential of the PDA cell lines was
determined using a modified two-chamber invasion assay, as
previously described .
In vivo experiments
ShCTRL or shENO1 CFPAC-1 cell lines were harvested,
washed three times, and resuspended in PBS.
NODSCID IL2Rgammanull (NSG) mice (provided by the
animal facility of the Molecular Biotechnology Center,
University of Turin, Italy) were injected into the tail vein
(i.v.) with 1×105 PDA cells (in 0.1 ml PBS). After 28 days,
mice were euthanized, necropsied, and examined for the
presence of tumor masses. For the in vivo experiments,
five mice were used in each group.
Tissue samples and histopathology
Tumor masses and main organs of mice were fixed in
4% (v/v) neutral-buffered formalin (Sigma-Aldrich)
overnight, transferred to 70% ethanol, followed by
paraffinembedding. For histological analysis, 5-μm
formalinfixed paraffin-embedded tissue sections were cut and
stained with hematoxylin-eosin. Tumor/normal tissue
ratios were evaluated with ImageJ software.
The Student’s t test (GraphPad Prism 5 Software, San
Diego, CA) was used to evaluate statistically significant
differences in in vitro and in vivo tests. Values were
expressed as mean ± SEM.
Altered expression of adhesion and cytoskeletal proteins
in shENO1 PDA cells
The CFPAC-1 PDA cell line was silenced with a
lentivirus that delivered a short hairpin RNA targeting ENO1
3’UTR (shENO1), or a scrambled shRNA (shCTRL) as a
control . Previous LC-MS/MS semi-quantitative
proteomic analysis using LTQ-Orbitrap on shENO1
CFPAC-1 cells showed significant alterations in the
expression of 17 proteins involved in cell adhesion and
cytoskeleton organization . Four of these proteins
[actin related protein 2/3 complex subunit 4 isoform a
(ARPC4), capping protein actin filament muscle Z-line
alpha 2 (CAPZA2), secreted phosphoprotein 1 isoform a
(SPP1 also named Osteopontin), and breast cancer
antiestrogen resistance 1 (BCAR1 also named p130cas)]
were upregulated, and 13 [AHNAK nucleoprotein
isoform 1 (AHNAK), anterior gradient protein 2 (AGR2),
catenin, delta 1 isoform 1ABC (CTNND1), hypothetical
protein LOC64855 isoform 2 (MINERVA), Galectin 3
(LGALS3), catenin alpha 1 (CTNNA1), integrin alpha v
isoform 1 precursor (ITGAV), Galectin 4 (LGALS4),
Golgi apparatus protein 1 isoform 1 (GLG1), mucin 5AC
(MUC5AC), serine or cysteine proteinase inhibitor clade
B ovalbumin member 5 (SERPINb5), PDZ and LIM
domain 1 (PDLIM1), and cysteine-rich protein 1 intestinal
(CRIP1)] were downregulated .
Herein, we studied whether the previously observed
protein modulation also occurred at the RNA level.
Quantitative real-time PCR analysis in shENO1
CFPAC1 cells indicated that, of the four upregulated proteins,
only BCAR1 (p130cas) showed a significant increase in
mRNA expression, while the other three proteins had
unchanged mRNA expression (Fig. 1). Among the 13
proteins that were downregulated after ENO1 silencing,
the expression of mRNA was significantly reduced in
nine of them, namely, AGR2, MINERVA, LGALS3,
CTNNA1, ITGAV, LGALS4, SERPINSb5, PDLM1, and
CRIP1. The mRNA expression was unchanged in three
of the remaining four proteins (AHNAH, CTNND1, and
GLG1) or was upregulated (MUC5AC) (Fig. 1).
Morphological and nanostructural modifications in
shENO1 PDA cells
Semi-quantitative proteomic and mRNA expression
analysis of PDA cells concordantly revealed downregulation
of nine cell-ECM adhesion-related proteins after ENO1
silencing (Ref.  and Fig. 1), and we therefore
investigated the impact of ENO1 silencing on the actin
cytoskeleton organization and morphology, by confocal
analysis. The majority of ENO1 protein (green
fluorescence) in shCTRL cells is localized to the cytoplasm
(Fig. 2a left panels), while after ENO1 silencing, green
fluorescence was lost, as expected (Fig. 2a right panels).
Analyzing actin organization (red fluorescence) showed
a loss of fluorescence close to the cell membrane in
shENO1 cells, with a concomitant increase of
cytoplasmic actin with a perinuclear accumulation. Additional
experiments performed with lower cell confluence and
with images taken at the level of the apical surface
showed that shCTRL cells possess well-defined actin
filaments (red fluorescence Fig. 2b upper panel), while
Fig. 1 mRNA expression of modulated proteins in CFPAC-1 shENO1
cells. Using real-time PCR, mRNA expression of different proteins was
investigated in CFPAC-1 shENO1 cells. Values are expressed as
relative expression compared to control cells. A representative of
three independent experiments is shown. Data are mean ± SEM.
*p < 0.05, **p < 0.01, ***p < 0.001 relative to control cells
shENO1 cells showed a loss of cytoskeleton organization
and orientation (red fluorescence Fig. 2b lower panel).
These confocal microscopy results were then combined
with cell surface analysis in atomic force microscopy
(AFM). Three-dimensional AFM highlighted that
cellcell junctions were impaired in shENO1 cells (Fig. 2c
lower panels) compared to shCTRL cells (Fig. 2c upper
panels). The 2-D AFM analysis of the cell surface
showed that shCTRL cells had a smooth and intact cell
surface and cell membrane ultrastructural components
were uniformly distributed (Fig. 2d upper panels). By
contrast, shENO1 cells displayed an altered surface
morphology with more evident membrane ultrastructure
(Fig. 2d lower panels). Quantification of the
nanostructural parameters revealed an increase in the cell surface
roughness (Fig. 2e) of shENO1 cells, in line with the
modification of the expression of proteins involved in
the remodeling of the cytoskeleton and adhesion (Ref.
 and Fig. 1).
shENO1 PDA cells display reduced adhesion to
fibronectin and collagens and increased adhesion to
As we showed that the proteins involved in ECM cell
adhesion were downregulated in shENO1 cells, we
evaluated the adhesive ability of shCTRL or shENO1 PDA cells
on fibronectin (FN), collagen I (Col-I), collagen IV
(ColIV), and vitronectin (VN). Compared to shCTRL cells,
shENO1 PDA cells (CFPAC-1, PT45 and T3M4) showed a
significantly lower adhesion to FN, Col-I, and Col-IV, and a
greater adhesion to VN (Fig. 3 and Additional file 1: Figure
S1a–b). We then evaluated the expression levels of alpha 5/
beta 1 and alpha v/beta 3 complexes, the integrins most
involved in binding on these extracellular matrices, in
shCTRL and shENO1 cells. Alpha 5 integrin was strongly
upregulated by shENO1, both at the mRNA (Fig. 4a) and
protein levels (Fig. 4b), while the mRNA, protein, and cell
surface levels of beta 1 were unchanged (Fig. 4a–c). mRNA
and protein levels of alpha v and beta 3 integrins were
decreased by shENO1 (Fig. 1, Fig. 4a and b, respectively).
Consistently, the surface expression of the complex alpha
v/beta 3, evaluated by a specific Ab that recognized the
whole complex, was decreased on shENO1 cells compared
to control cells (Fig. 4c). To identify the mechanism
involved in the increased VN adhesion mediated by ENO1
silencing, we also analyzed the expression of the other VN
receptor alpha IIb/beta 3 integrin. However, no surface
expression of this complex was detected in either shENO1 or
control cells (Fig. 4c). To understand the involvement of
beta 3 integrin in the adhesion to VN, a specific anti-beta 3
Ab was employed. In beta 3-expressing shCTRL cells, the
anti-beta 3 Ab significantly decreased the adhesion to VN,
while in shENO1 cells that did not express beta 3, it had no
effect (Fig. 4d). The increase in adhesion to VN in shENO1
Fig. 2 Morphological analysis and roughness measurements of shENO1 cells. a shCTRL and shENO1 CFPAC-1 cells stained for nuclei (DAPI, blue fluorescence),
actin (Phalloidin-TRITC, red fluorescence), and ENO1 (anti-ENO1 mAb followed by anti-mouse FITC, green fluorescence). Panels represent images taken at the
level of nuclei. b Images taken at the level of the cell surface. c Three-dimensional topographic images of shCTRL and shENO1 CFPAC-1 performed through
the AFM technique (three representative images for each group) (scan size: 50 μm; Z scale 4 μm). d Topographic images of shCTRL and shENO1 CFPAC-1
performed through the AFM technique. Left panels: height parameter; central panels: deflection parameter; right panels: magnification of deflection
panel. e Histograms represent roughness analysis. A representative of three independent experiments is shown. Data are mean ± SEM. ***p < 0.001
relative to control cells
cells with a concomitantly reduced expression of the major
integrins usually involved in its binding suggested the
involvement of other receptors. As uPAR binds VN through
the Somatomedin B (SMB) domain [31, 32], its expression
in shENO1 cells was evaluated. Quantitative PCR (Fig. 4a)
and western blot (Fig. 4b) analysis showed that uPAR
expression was markedly increased in shENO1 cells. These
data indicated that the lack of ENO1 caused a decrease in
the expression of the main integrins affecting cell adhesion.
However, shENO1 cells maintained the ability to bind VN
by upregulating uPAR.
Analysis of signaling pathways in shENO1 PDA cells
It is known that uPAR, despite lacking a cytosolic
domain, activates an intracellular signaling pathway
through the interaction with beta 1 and beta 3 integrins,
independently of their interaction with VN [33–35]. As
beta 1 expression was unchanged at the cell surface
of shENO1 cells (Fig. 4 b–c), and it can trigger
activation of the ERK1-2/RAC pathway [33, 36], the
effect of ENO1 silencing on this pathway was analyzed.
Western blot analysis revealed a strong increase in
activation of both ERK1-2 (Fig. 5a) and RAC (Fig. 5b)
Fig. 3 Adhesion ability of ENO1-silenced PDA cells. Adhesive potential of shENO1 and control CFPAC-1 cells was evaluated by culturing cells for
1 h on fibronectin (a), collagen I (b), collagen IV (c), and vitronectin (d). Adherent cells were fixed with 2% glutaraldehyde in PBS and visualized
by staining with crystal violet. For quantitative analysis, cells were treated with 10% acetic acid and elutes were read with a microplate reader at a
wavelength of 570 nm. Results are expressed as ΔOD (optical density) units = (OD substrate adherent cells)–(OD plastic adherent cells). A representative
of three independent experiments is shown. Data are mean ± SEM. **p < 0.01, ***p < 0.001 relative to control cells
Fig. 4 ENO1-silencing modulates ECM-receptor expression. a Using quantitative PCR, mRNA coding for different proteins was investigated
in shENO1 CFPAC-1 cells. Values are expressed as relative expression compared to control cells. b Western blot analysis was carried out to
investigate alpha 5, beta 1, alpha v, beta 3, and uPAR expression on total lysates of shCTRL and shENO1 cells. c To determine surface expression of integrins,
shENO1, or shCTRL cells were incubated with primary antibodies (gray peak) against beta 1, alpha v/beta 3, alpha IIb/beta 3, and or isotype-matched control
antibody (empty peak) and analyzed by flow cytometry. d The adhesion ability of shENO1 and shCTRL cells on vitronectin was evaluated in the presence of
anti-beta 3 Ab. Adherent cells were fixed with 2% glutaraldehyde in PBS and visualized by staining with crystal violet. For quantitative analysis, cells were
treated with 10% acetic acid and elutes were read with a microplate reader at a wavelength of 570 nm. Results are expressed as OD, optical density units.
A representative of three independent experiments is shown. Data are mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 relative to control cells
Fig. 5 Analysis of uPAR/integrins pathways. Western blot analysis on total lysates of shENO1 and shCTRL CFPAC-1 cells was carried out to investigate
levels of (a) phospho- and total ERK1-2, (b) activated and total RAC, (c) phospho- and total Src, (d) phospho- and total p38MAPK, (e) phospho- and
total AKT, (f) phospho- and total FAK and (g) phospho- and total Paxillin. Histograms represent the ratios between the phosphorylated and total form
of each protein. A representative of three independent experiments is shown
in shENO1 cells compared to control cells. To better
clarify the effect of ENO1 silencing on
integrindependent signaling cascades, other key proteins such
as the Src, p38MAPK, AKT, FAK, and Paxillin were
evaluated. In ENO1-silenced CFPAC-1 cells, the
phosphorylation of Src was increased (Fig. 5c) whereas
phosphorylation of p38MAPK were downregulated
(Fig. 5d). No significant difference was observed in
AKT, FAK, and Paxillin (Pax) phosphorylation
(Fig. 5e–g). Thus, ENO1 silencing increases Src
activation supporting downstream signals via ERK1-2/
uPAR blockade in shENO1 cells causes a reduction of ROS
and inhibition of cell senescence
Our previous work demonstrated that ENO1 silencing
increased reactive oxygen species (ROS) mainly
generated through the sorbitol and NADPH oxidase pathways,
which affect cancer cell growth and induce senescence
. As ERK1-2/RAC activation leads to an increase of
ROS and senescence , we investigated if uPAR is also
involved in these phenomena. ROS were analyzed by
measuring intracellular 5-(and-6)-chloromethyl-
2′,7′dichorodihydro-fluorescein diacetate (DCFDA)
fluorescence. Treatment with the anti-uPAR antibody blocked
the binding of uPAR to beta 1 integrin and specifically
reduced the production of ROS in shENO1 cells, but not
in shCTRL control cells (Fig. 6a).
As uPAR directly binds vitronectin, we observed that
the anti-uPAR blocking antibody inhibited senescence of
shENO1 cells plated on vitronectin (Fig. 6b upper
panel), but not that of shENO1 cells plated on collagen I
(Fig. 6b lower panel) suggesting that the senescence can
be induced by VN/uPAR downstream effects. The
antiuPAR blocking antibody slightly increased the apoptosis
of shENO1 PDA cells (Annexin V positive cells Fig. 6c).
ENO1 silencing impairs in vitro and in vivo PDA cell
migration and invasion
Considering the cytoskeleton and integrin pathway
alterations in shENO1 cells, we investigated their migration
and invasiveness by wound-healing scratch and Matrigel
cell invasion assays, respectively. While control cells
resulted in more than 60–80% of the wound being closed at
15 h, and 100% after 24 h, there was no wound-healing
evident on the shENO1 cell lines (CFPAC-1, PT45, and
T3M4), even after 24 h (Fig. 7a and Additional file 1:
Figure S1c). Cell invasion through Matrigel also
demonstrated a significantly lower ability of shENO1 cell lines to
invade compared to control cells (Fig. 7b and Additional
file 1: Figure S1d).
To assess the effect of ENO1 silencing in metastasis
formation in vivo, shENO1 or shCTRL CFPAC-1 cells
were injected intravenously into NSG mice. After
28 days following injection, mice were sacrificed and
lungs were excised and checked for the presence of
metastasis. Post-mortem observations confirmed that
injection with shENO1 cells resulted in a significant
reduction of the tumor area in the lungs compared to
mice injected with control cells (Fig. 7c). These data
confirmed that changes in the adhesion, migration, and
invasion ability observed in vitro in shENO1 cells
markedly compromised their ability to spread and form
Fig. 6 Analysis of reactive oxygen species (ROS) production, senescence, and apoptosis in shENO1 cells lines after anti-uPAR Ab treatment. a Analysis of
ROS concentration measured by the DCFDA-AM assay was evaluated in shENO cells and control cells in the presence or absence of anti-uPAR antibody.
b Senescence-associated β-galactosidase staining. Senescent shENO and shCTRL cells were colored blue upon X-gal staining at pH 6, with
or without treatment with anti-uPAR antibody. One representative out of three independent experiments is shown. The graph represents
the percentage of X-Gal positive cells with respect to the total number of cells. c Dot plot of shENO1 cells apoptotic cells in the presence
or absence of anti-uPAR antibody. Apoptotic cells were evaluated as early apoptotic (AnxV pos/ PI negative) + late apoptotic cells (AnxV
pos/ PI positive). Data are mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 relative to control cells
Fig. 7 ENO1 silencing affects in vitro and in vivo cell migration and
invasion. a Migration ability was evaluated in terms of capacity to
close the wound of shENO1 CFPAC-1 cells compared to shCTRL
control cells. Representative images are shown for each condition. b
Invasive potential of shENO1 and shCTRL cells were tested through
a Matrigel invasion assay, after 48 h of culture. For quantitative analysis,
invasive cells were fixed, stained with crystal violet, treated with acetic
acid and elutes were read at a wavelength of 570 nm. Results are
expressed as OD. c Lung metastatic area was evaluated after 28 days
from i.v. injection of shCTRL or shENO1 CFPAC-1 cells in the tail vein of
NSG mice. The histogram represents the percentage of metastatic area
compared to the total lung (left panel). Representative images of the
lung are shown for each group of mice (right panels). For the in vivo
experiments, five mice per group were used. A representative of three
independent experiments is shown. Data are mean ± SEM. *p < 0.05
relative to control cells
metastasis in vivo, demonstrating that ENO1 may exert
a crucial role in invasion and metastasis.
In our previous report, we demonstrated that ENO1 cell
surface expression is important for plasminogen-dependent
invasion, and that targeting of ENO1 with a monoclonal
antibody inhibits the invasiveness of pancreatic cancer cells
. These data correlate well with previous observations
in follicular thyroid carcinoma , glioma , non-small
cell lung cancer , and endometrial cancer , again
supporting the emerging role of ENO1 as a promising
target for cancer treatment. However, although blocking
surface ENO1 impaired the ability of PDA cells to migrate
in vitro and form more metastasis in vivo , little is
known about the role of ENO1 in regulating cell-cell and
cell-matrix contacts. Proteomic analysis of silenced ENO1
in PDA cells revealed a profound modification in their
metabolism, which was associated with an increase of
oxidative stress and senescence . In addition, although
silenced PDA cells also displayed alterations in many
molecules involved in adhesion, as well as cytoskeletal proteins,
this study aimed to investigate more thoroughly the role of
ENO1—a multifunctional protein involved in cell-matrix
adhesion, motility and migration, invasion and metastasis
in vivo, as well as in survival and senescence.
By employing a combination of confocal and
AFMassisted nanostructural approaches, we investigated the
phenotype and morphology of the PDA cells in the
presence and absence of ENO1. Profound morphological
changes in shENO1 PDA cells were observed, due to the
modification of the cytoskeleton organization. Control
cells exhibited smooth topography, while the shENO1
cells displayed a rough surface. The increase in surface
roughness was consistent with the topographical changes
observed with the AFM images. Cell nanostructural
parameters, such as elasticity or roughness, may reflect a
reorganization of the actin cytoskeleton, which in turn
affects cell growth, morphology, cell-cell interactions,
cytoskeleton organization, and interactions with the ECM
. It is known that ENO1 knockdown induces a
dramatic increase of the sensitivity to microtubule-targeted
drugs (e.g., taxanes and vincristine) in different tumor cell
lines, due to ENO1-tubulin interactions , suggesting a
role for ENO1 in modulating the cytoskeletal network.
Consistently, we demonstrated that ENO1 critically
contributed to the organization of the actin cytoskeleton.
Indeed, in control cells, actin was organized into filaments
that were mostly distributed close to the cell surface, while
in shENO1 cells, the organization of the actin filaments
was lacking, and actin prevalently relocalized close to the
nuclei. This actin modification suggested a profound
reorganization of cytoskeleton regulatory proteins,
suggesting that ENO1 silencing reduced the ability of PDA
cells to adhere to ECM and migrate. This is in agreement
with our previous proteomic analysis, which showed that
ENO1 silencing downregulated many proteins involved in
motility pathways .
Consistent with the proteomic data, shENO1 PDA cells
showed a decreased adhesion to FN and Cols, and a
reduction in migration and invasion. Of the proteins
downregulated after ENO1-silencing, we highlight alpha v/beta
3 integrin, a crucial protein involved in spreading and
metastasis, the increased expression of which correlates with
a poor clinical outcome in PDA [21–23]. FN is recognized
by either alpha 5/beta 1 or alpha v/beta 3 complexes,
which cooperate in promoting cellular attachment and
spreading . Our data show that ENO silencing
downregulates alpha v/beta 3 but increases alpha 5/beta 1
expression. Considering that alpha 5/beta 1 determines
adhesion strength through its binding to FN, while alpha
v/beta 3 mediates reinforcement of the adhesion through
its connection with the actin cytoskeleton [42, 43], we
speculate that in shENO1 cells, while the alpha 5/beta 1
complex begins the adhesion process, there is a reduced
adherence to FN due to the lack of reinforcement signals
controlled by the alpha v/beta 3 complex. Surprisingly,
ENO1 silencing is also associated with an increase of
adhesion to VN. In cancer, VN interacts with different
members of the integrin family (alpha v/beta 1, alpha v/
beta 3, alpha v/beta 5, and alpha IIb/beta 3) through the
RGD motif [44, 45]. As alpha v and beta 3 subunits were
shown to be downregulated in shENO1 cells, none of the
abovementioned complexes can be considered to be
responsible for the increased adhesion of shENO1 cells to
VN. This suggests that a non-integrin receptor is involved
in the binding of VN. As uPAR binds VN through a
different binding site from that of integrins [34, 46, 47],
we hypothesized that uPAR plays a major role in VN
binding in shENO1 cells.
uPAR expression is elevated in many human cancers, in
which it frequently indicates poor prognosis . uPAR
regulates proteolysis by binding to the extracellular
protease urokinase-type plasminogen activator (uPA)  and
also activates many intracellular signaling pathways .
Exploiting the above functions, uPAR regulates important
functions such as cell migration, proliferation, and survival,
and thus makes it an attractive therapeutic target in various
different types of cancer . Here, we observed the
upregulation of uPAR in shENO1 cells, which have a reduced
ability to invade and form metastases, and an increased
senescence, suggesting that uPAR may contribute in a
different way in this setting. uPAR, lacks a cytosolic domain,
and thus signals through its association with integrins,
which can also be independent of direct integrin/matrix
interactions, in a ligand-independent manner, to promote
migration . The major downstream uPAR/integrin
signaling (especially beta 1 and beta 3) involves the activation
of Src, PI3K/AKT, and MEK/ERK1-2 pathways . In
shENO1 PDA cells we observed the activation of Src. The
effect of increased Src activity in cells is pleiotropic . In
particular, cells with activated Src are characterized by a
loss of actin reorganization and reduced cell-ECM
adhesion , phenomena that perfectly match our
In NSCLC, the downregulation of ENO1 decreased
proliferation, migration, and invasion through a FAK-mediated
PI3K/AKT pathway . Clustering of integrins activates
FAK results in the formation of a complex with Src, and
increased phosphorylation of the targets of the FAK-Src
complex, such as paxillin . In pancreatic cancer we did
not observe an altered phosphorylation of FAK and Paxillin
as well as AKT. Instead, in our study, we observed that
ENO1 silencing leads to uPAR overexpression that, in turn,
triggers Src and ERK1-2 activation, concomitantly with an
inactivation of p38MAPK. The activation of ERK1-2 can
promote senescence, in accordance with Cagnol et al. .
Conversely, activation of p38MAPK can promote apoptosis
in cancer cells, including pancreatic cancer . We
observed that shENO1 cells, due to the decrease in
phosphorylation of p38MAPK concurrently with ERK1-2 activation,
slightly inhibit PDA cell apoptosis and favor senescence, in
line with our previously reported results .
ERK1-2 activation, due to the uPAR-beta 1 integrin
interaction is required for RAC activation [36, 55–59]. RAC is a
downstream signaling molecule of beta 1 and contributes
to the regulation of actin cytoskeleton dynamics, adhesion,
and migration and induces cellular reactive oxygen species
(ROS) through NAPDH oxidase activation . Expression
of the constitutively active RAC1 mutant induces cell cycle
arrest, apoptosis, and senescence . We have previously
demonstrated that ENO1 silencing induces ROS, mainly
through the sorbitol and NADPH oxidase pathway and
senescence . Here we demonstrate that, in the absence
of ENO1, the upregulation of uPAR leads to an increased
activation of the ERK1-2/RAC pathway, which contributes
to ROS generation and induces PDA cell senescence, rather
than an invasive phenotype. Moreover, the anti-uPAR
antibody prevented ROS production and senescence although
PDA cell apoptosis was only slightly promoted. These
results suggest that a combinatory strategy to simultaneously
target ENO1 and uPAR could be effective to inhibit PDA
tumor progression and invasion.
Our study has shown, by in vitro and in vivo experiments,
that ENO1 silencing can inhibit adhesion, invasion, and
metastasis in PDA cells, due to changes in actin
cytoskeleton organization, adhesion proteins, and integrin profile
expression. ENO1 silencing had a major impact on the
alpha v/beta 3 integrin, which accounts for the inability of
ENO1-silenced cells to adhere to the ECM matrix and
promote PDA invasion. We have reported that, in the absence
of ENO1, the upregulation of uPAR does not promote an
increase of migration or invasion. These data show that
there is an interplay of ENO1 with integrins and uPAR,
which critically controls PDA progression.
Additional file 1: Table S1. Oligonucleotide primer sequences for
SybrGreen qRT-PCR. Figure S1a–d Adhesion, migration and invasion
ability of PT45 and T3M4 shENO1 PDA cells. Adhesive potential of
shENO1 PT45 (a) and shENO1 T3M4 (b) compared to relative shCTRL
control cells was evaluated by culturing cells for 1 h on FN, Col-I, Col-IV
and VN. Adherent cells were fixed with 2% glutaraldehyde in PBS and
visualized by staining with crystal violet. For quantitative analysis, cells
were treated with 10% acetic acid and elutes were read with a
microplate reader at a wavelength of 570 nm. Results are expressed as Δ ⋿
OD (Optical Density) units = (OD substrate adherent cells) – (OD plastic
adherent cells). (c) Migration ability was evaluated in terms of ability to close
the wound with shENO1 PT45 and shENO1 T3M4 cells compared to shCTRL
control cells. Representative images are shown for each condition. (d) Invasive
potential of shENO1 PT45 and shENO1 T3M4 were tested by a Matrigel
invasion assay, after 48 h of culture. For quantitative analysis, invasive cells
were fixed, stained with crystal violet, treated with acetic acid and elutes were
read at a wavelength of 570 nm. Data are mean ± SEM of at least three
independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 (DOCX 326 kb)
Ab: Antibody; AFM: Atomic force microscopy; AGR2: Anterior gradient protein 2;
AHNAK: AHNAK nucleoprotein isoform 1; AnxV: Annexin V; APC: Allophycocyanin;
ARPC4: Actin related protein 2/3 complex subunit 4 isoform a; ATP: Adenosine
triphosphate; BCAR1: Breast cancer anti-estrogen resistance 1; CAPZA2: Capping
protein actin filament muscle Z-line alpha 2; CAT: Confocal–atomic force
microscopy–total internal reflection fluorescence; Col: Collagen; CRIP1:
Cysteinerich protein 1 intestinal; CTNNA1: Catenin alpha 1; CTNND1: Catenin, delta 1
isoform 1ABC; ECM: Extracellular matrix; ENO1: Alpha-enolase; ERK1-2: Extracellular
signal–regulated kinases 1–2; FAK: Focal adhesion kinases; FN: Fibronectin;
GLG1: Golgi apparatus protein 1 isoform 1; GST: Glutathione S-transferase;
HRP: Horseradish peroxidase; IgG: Immunoglobulin G; ITGAV: Integrin alpha v
isoform 1 precursor; LC-MS/MS: Liquid chromatography-mass/mass spectrometry;
LGALS3: Galectin 3; LGALS4: Galectin 4; MAPK: Mitogen-activated protein kinase;
MINERVA: Hypothetical protein LOC64855 isoform 2; MUC5AC: Mucin
5AC; NSG: NOD/SCID gamma; Pax: Paxillin; PBS: Phosphate-buffered
saline; PCR: Polymerase chain reaction; PDA: Pancreatic ductal adenocarcinoma;
PDLIM1: PDZ and LIM domain 1; PI: Propidium iodide; RGD: Arg-Gly-Asp;
ROS: Reactive oxygen species; SA-beta-Gal: Senescence associated
betaGalactosidase; SEM: Standard error of the mean; SERPINb5: Serine or cysteine
proteinase inhibitor clade B ovalbumin member 5; shCTRL: Control cell line;
shENO1: ENO1-silenced cell line; shRNA: Short hairpin RNA; SMB: Somatomedin
B; SPP1: Secreted phosphoprotein 1 isoform a; TRITC: Tetramethylrhodamine B
isothiocyanate; uPAR: Urokinase plasminogen activator receptor; VN: Vitronectin
This study was supported by the Associazione Italiana Ricerca sul Cancro (AIRC 5 x
mille no. 12182 and IG no. 15257); University of Turin-Progetti Ateneo
2014Compagnia di San Paolo (PC-METAIMMUNOTHER to FN and PANTHER to PC),
Italian Ministry of Health, Progetti Ricerca Finalizzata (RF-2013-02354892),
Fondazione Ricerca Molinette Onlus and Fondazione Nadia Valsecchi; MP
and MSC are recipients of fellowships funded by the Fondazione Ursula e
Giorgio Cytron. PD is a recipient of PI AIRC IG 2014N.15399. SL gratefully
acknowledges the financial support of the REA research grant no.
PITNGA-2012-316549 (IT LIVER) from the People Programme (Marie Curie Actions) of
the European Union’s Seventh Framework Programme (FP7/2007-2013). Financial
support from MAAT-Molecular NAnotechnology for HeAlth and EnvironmenT
Project (number PON02_00563_3316357–CUP B31C12001230005) is also gratefully
MP designed and performed most of the in vitro and in vivo experiments,
analyzed results, generated figures and tables, and wrote the manuscript. SB
performed in vitro and in vivo experiments, analyzed data, and wrote the
manuscript. MSC, SFB, and SBul performed in vitro experiments and analyzed
data. MCap performed proteomics experiments and analyzed data. MCas
and SL designed and performed experiments with the CAT microscope and
analyzed data. JC performed pull down and immunoblot experiments for
activated RAC. PD, FDM, and PN provided reagents, discussed experimental
design for the integrin studies and analyzed data. CR performed ROS
measurement experiments and analyzed data. PC designed experiments,
analyzed data, and revised the manuscript. FN designed experiments,
supervised the study, analyzed data, and wrote the manuscript. All authors
read and approved the final manuscript.
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