Background
Pancreatic ductal adenocarcinoma (PDA) is one of the most lethal forms of cancer with a 5-year survival rate of less than 8% [
1]. 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 (ENO1) [
8].
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 [
12]. 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 adhesion-dependent growth and invasion of tumor cells and the integrin alpha v/beta 3 has been reported as a mediator of anchorage independence [
21], 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.
Methods
Cell culture
Human pancreatic cancer cell lines used were: CFPAC-1 (from ECACC), PT45, and T3M4. Cell lines were cultured in DMEM (Lonza, Milan, Italy) supplemented with 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 [
12].
Quantitative PCR
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).
CAT microscopy
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 confocal LSM.
AFM imaging
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 [
28]. 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 μm
2 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.
Confocal microscopy
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).
Adhesion assay
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 [
29]. 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 [
30]) 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 anti-human 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× Laemmli-reducing sample buffer and immunoblotted for RAC1.
ROS measurement
PDA cells were cultured for 24 h in presence or absence of 10 μg/ml anti-uPAR antibody (clone 3C6, Sigma-Aldrich). ROS measurement was performed as previously described [
19].
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 × 10
4 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, Sigma-Aldrich). Images were taken at microscope with ×10 objective. The SA-β-galactosidase activity was analyzed as previously described [
19].
Apoptosis assay
PDA cells were cultured for 48 h in presence or absence of 10 μg/ml anti-uPAR antibody (clone 3C6, Sigma-Aldrich), 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 [
12].
In vivo experiments
ShCTRL or shENO1 CFPAC-1 cell lines were harvested, washed three times, and resuspended in PBS. NOD-SCID 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 paraffin-embedding. For histological analysis, 5-μm formalin-fixed paraffin-embedded tissue sections were cut and stained with hematoxylin-eosin. Tumor/normal tissue ratios were evaluated with ImageJ software.
Statistical analysis
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.
Discussion
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 [
12]. These data correlate well with previous observations in follicular thyroid carcinoma [
38], glioma [
13], non-small cell lung cancer [
14], and endometrial cancer [
15], 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 [
12], 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 [
19]. 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 AFM-assisted 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 [
39]. 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 [
40], 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 [
19].
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 [
41]. 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 [
48]. uPAR regulates proteolysis by binding to the extracellular protease urokinase-type plasminogen activator (uPA) [
49] and also activates many intracellular signaling pathways [
50]. 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 [
36]. 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 [
34]. The major downstream uPAR/integrin signaling (especially beta 1 and beta 3) involves the activation of Src, PI3K/AKT, and MEK/ERK1-2 pathways [
50]. In shENO1 PDA cells we observed the activation of Src. The effect of increased Src activity in cells is pleiotropic [
51]. In particular, cells with activated Src are characterized by a loss of actin reorganization and reduced cell-ECM adhesion [
51], phenomena that perfectly match our observations.
In NSCLC, the downregulation of ENO1 decreased proliferation, migration, and invasion through a FAK-mediated PI3K/AKT pathway [
14]. 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 [
52]. 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. [
53]. Conversely, activation of p38MAPK can promote apoptosis in cancer cells, including pancreatic cancer [
54]
. 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 [
19].
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 [
37]. Expression of the constitutively active RAC1 mutant induces cell cycle arrest, apoptosis, and senescence [
37]. We have previously demonstrated that ENO1 silencing induces ROS, mainly through the sorbitol and NADPH oxidase pathway and senescence [
19]. 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.
Acknowledgements
We thank Dr. Radhika Srinivasan for helpful discussion and critical reading of the manuscript, and Roberta Curto for the in vivo experiments.