E XP ER I ME NTAL C E LL RE S E ARCH
24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41Q1 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83
] (]]]]) ]]]–]]]
Available online at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/yexcr
Research Article
Induction of pancreatic cancer cell migration by an autocrine epidermal growth factor receptor activation Anna-Maria Stocka, Stephan A Hahnb, Gabriele Troosta, Bernd Niggemanna, Kurt S Zänkera, Frank Entschladena,n a
Institute of Immunology and Experimental Oncology, Centre for Biomedical Education and Research (ZBAF), Witten/Herdecke University, 58448 Witten, Germany b Department of Molecular Gastroenterological Oncology, Centre of Clinical Research, Ruhr-University of Bochum, 44780 Bochum, Germany
article information
abstract
Article Chronology:
Pancreatic cancer is characterized by aggressive local invasion and early metastasis formation. Active
Received 17 February 2014
migration of the pancreatic cancer cells is essential for these processes. We have shown previously that
Received in revised form
the pancreatic cancer cells lines CFPAC1 and IMIM-PC2 show high migratory activity, and we have
24 April 2014
investigated herein the reason for this observation. Cell migration was assessed using a three-
Accepted 28 April 2014
dimensional, collagen-based assay and computer-assisted cell tracking. The expression of receptor tyrosine kinases was determined by flow-cytometry and cytokine release was measured by an
Keywords: Pancreatic cancer Metastasis Cell migration Receptor tyrosine kinases Epidermal growth factor
enzyme-linked immunoassay. Receptor function was blocked by antibodies or pharmacological enzyme inhibitors. Both cells lines express the epidermal growth factor receptor (EGFR) as well as its familymember ErbB2 and the platelet-derived growth factor receptor (PDGFR)α, whereas only weak expression was detected for ErbB3 and no expression of PDGFRβ. Pharmacological inhibition of the EGFR or ErbB2 significantly reduced the migratory activity in both cell lines, as did an anti-EGFR antibody. Interestingly, combination of the latter with an anti-PDGFR antibody led to an even more pronounced reduction. Both cell lines release detectable amounts of EGF. Thus, the high migratory activity of the investigated pancreatic cancer cell lines is due to autocrine EGFR activation and possibly of other receptor tyrosine kinases. & 2014 Published by Elsevier Inc.
Introduction The difficult diagnosis of pancreatic cancer at a localized resectable stage makes the prognosis of patients with this disease extremely poor. Another reason of the poor prognosis is the early metastasis formation of the tumor to the regional lymph nodes and liver [1,2].
An essential prerequisite for metastasis formation is the active migration of malignant cells from the primary tumor via lymphatic or blood vessel routes. This complex process of cancer cell migration is regulated by many different signalling pathways as well as substances of various classes and origins [3]. The most important migrational regulators can be divided into two major groups: On the
Abbreviations: cAMP, cyclic AMP; EGF(R), epidermal growth factor (receptor); EMT, epithelial to mesenchymal transition; FITC, fluorescein isothiocyanate; FAK, focal adhesion kinase; GPCR, G protein-coupled receptor; mAb, monoclonal antibody; PI3, phosphatidyl-inositide 3; PLC, phospholipase C; HB-EGF, heparin-binding EGF-like growth factor; PDGF(R), platelet-derived growth factor (receptor); PKA/C, protein kinase A/C; TGF, transforming growth factor; VEGF(R), vascular endothelial growth factor(R) n
Corresponding author. Fax: þ49 2302 926 158. E-mail address: [email protected] (F. Entschladen).
http://dx.doi.org/10.1016/j.yexcr.2014.04.022 0014-4827/& 2014 Published by Elsevier Inc.
Please cite this article as: A.-M. Stock, et al., Induction of pancreatic cancer cell migration by an autocrine epidermal growth factor receptor activation, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.04.022
2
84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143
E XP E RI ME N TAL CE L L R ES E ARC H
one hand there are cytokines and growth factors, which regulate receptor kinases and related receptors with associated kinases [4,5]. The second group includes chemokines and neurotransmitters, which are able to activate members of the huge family of G protein coupled receptors (GPCRs) [6,7]. One example of these GPCR ligands is the stress catecholamine hormone norepinephrine. In previous in vitro cell migration studies we have observed that norepinephrine induces diverse effects on the migratory activity of different human tumor cells: some cell lines increase their migratory activity in response to norepinephrine, some do not show any significant norepinephrineinduced effects and others reduce their migratory activity after norepinephrine treatment [8–12]. The pancreatic cancer cell lines CFPAC1 and IMIM-PC2 are part of the last named group. The inhibitory effect of norepinephrine on the migration of these cells appears to be due to an imbalance of two cell signaling pathways. The protein kinase C/phospholipase C (PKC/ PLC) pathway is already activated in the absence of norepinephrine, leading to an activation of the motor protein non-muscle myosin II and thus to a high spontaneous migratory activity. Therefore, norepinephrine does not cause further activation of this PLC/PKC pathway, but instead activates the second cyclic AMP/protein kinase A (cAMP/PKA)-dependent pathway and finally leads to an inhibitory effect on pancreatic cancer cell migration [10]. The signalling pathway or signalling event, which is responsible for the constitutive PLCγ activation in pancreatic cancer cells, is not clear. We hypothesize that an autocrine activation of receptor tyrosine kinases might be the reason for the constitutive PLCγ activation. Ligand binding induces dimerization and autophosphorylation of receptor tyrosine kinases and results in phosphorylation of various SH2 domain-containing signal transduction molecules such as PLCγ, PI3-kinase, GAP and cSrc [13,14]. Examples of these receptor tyrosine kinases are the platelet-derived growth factor receptors α and β (PDGFRα and PDGFRβ) and the four members of the ErbB receptor family: the epidermal growth factor receptor (EGFR/ErbB1), HER2/neu (ErbB2), HER3 (ErbB3) and HER4 (ErbB4). The expression of the PDGF receptors and at least one of the members of the EGFR family has been proven in a large proportion of pancreatic cancers [15–17]. The activation of the different ErbB receptors can be induced by the following ligands: EGF, transforming growth factor alpha (TGF-α), amphiregulin, betacellulin, heparin-binding EGF-like growth factor (HB-EGF), epiregulin, epigen and neuregulins 1–6 [18,19]. Among these ligands, TGF-α, amphiregulin, betacellulin, epiregulin and EGF are specific activators of EGFR [20]. A study of Murphy et al. revealed that autocrine EGFR activation stimulates the proliferation of these cells [21]. Furthermore, different studies indicated that EGF and PDGF initiate the association of their respective receptors with PLCγ and induce the PLCγ phosphorylation [22,23]. In the context of these findings the present study focuses on the role of EGFR, ErbB2 and PDGFR in pancreatic cancer cell migration and provides EGF as an important autocrine factor for the regulation of the migratory activity of these cells.
Materials and methods Cell culture The pancreatic cancer cell line CFPAC1 was purchased from American Type Culture Collection (Manassas, VA, USA) and the pancreatic cancer cell line IMIM-PC2 was a gift from Francisco
] (]]]]) ]]]–]]]
X Real (Spanish National Cancer Research Centre, Madrid, Spain). The cells were grown in Dulbecco's Modified Eagle Medium (Sigma-Aldrich, Steinheim, Germany), supplemented with 10% fetal bovine serum (Biochrom AG, Berlin, Germany), 2% penicillin/streptomycin (Sigma-Aldrich), 1 mM sodium pyruvate (Biochrom) and 2 mM L-glutamine (PAA Laboratories GmbH, Linz, Austria) at 37 1C in a humidified atmosphere of 5% CO2. For the pancreatic cancer cell line CFPAC-1 reference STR data are available The GenomeLab Human STR Primer Set (Beckman Coulter, Krefeld, Germany) was used and analyzed on a CEQ8800 sequencer (Beckman Coulter) according to the manufacturer's protocol to control for the cell line identity. STR data were submitted to on-line verification tool of DSMZ (German Collection of Microorganisms and Cell Lines) to confirm identity (http://old. dsmz.de/human_and_animal_cell_lines/main.php?contentle ft_id=101). For IMIM-PC2, there is no reference available.
Cell migration assay The migratory activity of the two cell lines was analyzed as described in detail previously [24]. In short, 5 104 cells were mixed with carbonate-buffered collagen solution (Advanced BioMatrix, San Diego, CA, USA) containing minimal essential medium (Sigma-Aldrich) as well as one or two of the following substances: the EGFR kinase inhibitor AG1478 (300 nM, Merck KGaA, Darmstadt, Germany), EGF (100 ng/ml, Sigma-Aldrich), the ErbB2 inhibitor 4-(3-Phenoxyphenyl)-5-cyano-2H-1,2,3-triazole (10 mM, Merck KGaA), a neutralizing anti-EGFR antibody (10 mg/ml, Merck KGaA) and a neutralizing anti-PDGFRα antibody (10 mg/ml, Abcam, Cambridge, UK). After polymerization of the collagen, the migration of the cells was recorded by time-lapse video microscopy for 15 h. Subsequently two-dimensional projections of the paths of 30 randomly selected cells were digitized in 15 min intervals. The migratory activity was calculated for each step as the portion of cells, which was locomotory active. The average of the activity was calculated for all steps of the entire observation period. The graphs show the mean values and standard deviations of the migratory activity of at least three independent experiments. In minimum 90 cells were analyzed per sample. Statistically significant changes (po0.05) were calculated using Student's t-test (two-tailed and unpaired).
Flow cytometry A FacsCalibur flow cytometer (Becton Dickinson GmbH, Heidelberg, Germany) was used to investigate the expression of EGFR, ErbB2, ErbB3, PDGFRα and β in pancreatic cancer cells. 1 105 cells were fixed with 1% paraformaldehyde (SigmaAldrich), washed with 0.5% Triton X-100 (Sigma-Aldrich) and incubated with one of the following antibodies (Ab): anti-EGFR (Ab-1) mouse mAb (528) IgG2a (200 ng/ml, Merck KGaA), isotypic control IgG2a (1:250 dilution, Beckman Coulter, Marseille, France), anti-c-ErbB2/c-Neu (Ab-2) mouse mAb 9G6 (200 ng/ml, Merck KGaA), ErbB-3 (298) mouse monoclonal IgG1 (200 ng/ml, Santa Cruz Biotechnology, Heidelberg, Germany), isotypic control IgG1 (400 ng/ml, Beckman Coulter), PDGFRα (D13C6) XP™ rabbit mAb (1:50 dilution, New England Biolabs GmbH, Frankfurt), PDGFRβ (C82A3) rabbit mAb (1:50 dilution, New England Biolabs GmbH) or normal rabbit IgG (1:50 dilution, Santa Cruz Biotechnology). After 30 min incubation one of the following secondary
Please cite this article as: A.-M. Stock, et al., Induction of pancreatic cancer cell migration by an autocrine epidermal growth factor receptor activation, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.04.022
E X PE R IM EN TA L C ELL R E S EA RC H
In contrast to this high EGFR and ErbB2 expression, a faint expression of ErbB3 and PDGFRα was observed in both cell lines. The ErbB3 mean fluorescence intensity was 5.91 for the CFPAC1 and 2.22 for the IMIM-PC2 cells and the PDGFRα mean fluorescence intensity was 5.13 for the CFPAC1 and 7.62 for the IMIM-PC2 cells. Both cell lines contained no detectable amounts of PDGFRβ.
CFPAC1
Measurement of EGF The Quantikines ELISA Human EGF Immunoassay (R&D Systems Europe, Abingdon, UK) was used for the quantitative determination of human EGF concentrations in cell culture supernatants. The sample preparation and the measurement of the EGF concentrations were performed according to the manufacturer’s protocol. Cells (1 106 cells/ml) were seeded into 24-well-plates and incubated at 37 1C in a humidified atmosphere of 5% CO2. Samples were harvested every 24 h by centrifugation and stored at 20 1C. Every experiment was repeated three times. All standards and samples of one experiment were assayed in triplicate.
Results In previous studies we could show that the pancreatic cancer cell lines CFPAC1 and IMIM-PC2 developed a high spontaneous migratory activity after incorporation within a three-dimensional collagen matrix. Additionally, our results demonstrated that non-muscle myosin II seems to be constitutively activated via the PKC/PLC pathway in these cells [10]. According to these findings and the link between the PLCγ phosphorylation, EGFR and PDGFR [22,23], we analyzed the role of EGFR and PDGFR in the regulatory network of pancreatic cancer cell migration. Therefore, we investigated the expression of EGFR, ErbB2, ErbB3, PDGFRα and PDGFRβ on the cell surface of CFPAC1 and IMIM-PC2 cells by flow cytometric analysis (Fig. 1). In both cell lines the highest mean fluorescence intensity was detectable in the case of the EGF receptor: 24.18 for the CFPAC1 cells and 24.62 for the IMIM-PC2 cells. The mean fluorescence intensities of 23.62 (CFPAC1) and 11.87 (IMIM-PC2) indicated that the ErbB2 expression was slightly lower than the EGFR expression.
100
Locomoting cells (%)
antibodies was added: R-phycoerythrin-conjugated AffiniPure F (ab')2 fragment goat anti-mouse IgG (1:250 dilution, Dianova GmbH, Hamburg, Germany) or goat anti-rabbit IgG-FITC (8 mg/ml, Santa Cruz Biotechnology). All incubation steps were performed at room temperature. Additionally, the viability of the cells was assessed by propidium iodide staining and flow cytometry in order to exclude that a reduction of migratory activity might be due to cell death.
90 80 70 60 50 40 30 20 10 0
IMIM-PC2
Control
AG1478
ErbB2 Inhibitor
Control
AG1478
ErbB2 Inhibitor
100
Locomoting cells (%)
144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203
3
] (]]]]) ]]]–]]]
90 80 70 60 50 40 30 20 10 0
Fig. 2 – Migratory activity of pancreatic carcinoma cells in response to the EGFR kinase inhibitor AG1478 and the ErbB2 inhibitor 4-(3-Phenoxyphenyl)-5-cyano-2H-1,2,3-triazole. AG1478 was applied at a concentration of 300 nM and the ErbB2 inhibitor was applied at a concentration of 10 lM. The graphs show mean values and standard deviations of at least three independent experiments. Asterisks mark statistically significant changes (po0.05).
Fig. 1 – Expression of EGFR, ErbB2, ErbB3, PDGFRα and PDGFRβ on pancreatic cancer cells. Gray fields¼isotypic control antibody (ISO); black line ¼expression of EGFR, ErbB2 and ErbB3; MFI ¼mean fluorescence intensity. Please cite this article as: A.-M. Stock, et al., Induction of pancreatic cancer cell migration by an autocrine epidermal growth factor receptor activation, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.04.022
4
CFPAC1
100
Locomoting cells (%)
] (]]]]) ]]]–]]]
12
90 80
*
70
*
*
60
EGF (pg/ml)
CFPAC1
50 40 30 20 10
10 8 6 4
0
Control
EGF
EGFR EGFR antibody antibody & EGF
2 0
100
*
90
*
*
80 70
day 1
day 2
day 3
day 4
day 1
day 2
day 3
day 4
IMIM-PC2 12
60 50 40
EGF (pg/ml)
IMIM-PC2 Locomoting cells (%)
204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263
E XP E RI ME N TAL CE L L R ES E ARC H
30 20 10 0
Control
EGF
EGFR EGFR antibody antibody & EGF
Fig. 3 – Migratory activity of pancreatic carcinoma cells in response to EGF and an EGFR neutralizing antibody. EGF was applied at a concentration of 100 ng/ml and the EGFR neutralizing antibody was applied at a concentration of 10 lg/ml. The graphs show mean values and standard deviations of at least three independent experiments. Asterisks mark statistically significant changes (po0.05).
The flow cytometric analyses were followed by migratory experiments with AG1478 (300 nM; Fig. 2). This EGFR kinase inhibitor significantly decreased the spontaneous migratory activity of CFPAC1 cells from 47.4%71.6% to 33.8%74.4% (p¼ 0.004) and the activity of IMIM-PC2 cells from 48.4%76.8% to 29.6%75.2% (po0.001). On the basis of the high ErbB2 expression we also evaluated the effects of ErbB2 inhibition on pancreatic cancer cell migration (Fig. 2). The ErbB2 inhibitor 4-(3-Phenoxyphenyl)-5-cyano-2H-1,2,3-triazole (10 mM) significantly reduced the migratory activity of CFPAC1 cells from 47.4%71.6% to 28.3%74.0% (po0.001) and the migratory activity of IMIM-PC2 cells from 48.4%76.8% to 32.3%74.9% (p¼ 0.003). Furthermore, we found that treatment with 100 ng/ml of the EGFR ligand EGF augmented the spontaneous migratory activity of the CFPAC1 cells significantly (po0.001) from 40.3%72.3% to 51.9% 71.7% and the migratory activity of the IMIM-PC2 cells from 49.4% 71.8% to 64.2%710.0% (p¼0.027; Fig. 3). A concentration of 10 mg/ml of a neutralizing anti-EGFR antibody was able to inhibit this EGF-induced increased migratory activity. CFPAC1 cells showed a significant decrease (p¼ 0.002) of locomoting cells from 51.9%71.7% to 34.2%76.0% and IMIM-PC2 cells changed from 64.2%710.0% to 36.1%76.8% locomoting cells (p¼0.009). In addition to the decrease of the EGF-induced migratory activity, the neutralizing anti-EGFR antibody caused a significant reduction of the spontaneous migratory activity. In the case of the CFPAC1 cells the migratory activity diminished from 40.3%72.3% to 31.6%75.4% (p¼0.032) and in the IMIM-PC2 cells from 49.4%71.8% to 26.4%76.0% (po0.001). Due to the induction of the migratory activity by EGF, we used the Quantikines ELISA Human EGF Immunoassay to analyze the EGF levels in the cell culture supernatants over a period of four days (Fig. 4). The EGF concentration in the supernatant of the
10 8 6 4 2 0
Fig. 4 – Quantitative determination of human EGF concentrations in cell culture supernatants. The graphs show the mean values and the standard deviations of the EGF concentrations in cell culture supernatants after one, two, three and four days of three independent experiments. CFPAC1 cells increased from 0.9 pg/ml70.6 pg/ml after 24 hours to 8.1 pg/ml 71.9 pg/ml after four days and in the supernatant of the IMIM-PC2 cells from 0.8 pg/ml70.2 pg/ml to 12.0 pg/ml70.6 pg/ml. The measured amounts of secreted EGF have not been adjusted to the increase of the cell number during the incubation period. However, the cell growth has been analyzed previously [10]. The increase of secreted EGF by far exceeds the growth of the cells within the observation period of four days. Thus, there is an accumulation of EGF in the culture supernatant over time. According to the link between PLCγ phosphorylation, EGFR and PDGFR introduced before, we used a neutralizing PDGFRα antibody to investigate the influence of PDGFRα inhibition on the locomotory activity of pancreatic cancer cells (Fig. 5). In CFPAC1 cells this antibody did not lead to a significant effect on the migratory activity, whereas the percentage of locomoting IMIMPC2 cells significantly decreased from 30.9%75.4% to 21.1%72.3% (p¼ 0.020). A combination of the neutralizing PDGFRα and the neutralizing EGFR antibody reduced the migratory activity of CFPAC1 cells significantly from 26.2%74.8% to 18.7%73.8% (p¼ 0.049) and in IMIM-PC2 cells from 30.9%75.4% to 16.9% 72.2% (p¼ 0.013). Therefore, it seems likely that not only EGF but further growth factor receptors or cytokine receptors such as the PDGFR are involved in this autocrine stimulation of migration.
Discussion The development of autocrine loops makes tumor cells independent of exogenous growth factors, which is regarded as a hallmark
Please cite this article as: A.-M. Stock, et al., Induction of pancreatic cancer cell migration by an autocrine epidermal growth factor receptor activation, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.04.022
E X PE R IM EN TA L C ELL R E S EA RC H
CFPAC1 Locomoting cells (%)
100 90 80 70 60 50 40 30 20 10 0
IMIM-PC2
Control
PDGFR PDGFR& antibody EGFR antibodies
Control
PDGFR PDGFR& antibody EGFR antibodies
100
Locomoting cells (%)
264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323
90 80 70 60 50 40 30 20 10 0
Fig. 5 – Migratory activity of pancreatic carcinoma cells in response to a PDGFRα neutralizing antibody and an EGFR neutralizing antibody. Both antibodies were applied at a concentration of 10 lg/ml. The graphs show mean values and standard deviations of at least three independent experiments. Asterisks mark statistically significant changes (po0.05).
of cancer [25]. Autocrine induction of cancer cell migration by different growth factors has been described for various types of tumor cells. In 1999, the transforming growth factors α and β1 (TGFα and TGFβ1) were identified as autocrine acting motility factors in oral squamous carcinoma cells, which stimulate the autonomous migration of these cells [26]. Another autocrine TGFβ loop was observed in papillary thyroid carcinoma cells. In this case the oncogene BRAF induces TGFβ secretion as a part of a sodium/iodide symporter (NIS) repression mechanism. The secreted TGFβ leads to an increased epithelial to mesenchymal transition (EMT), migration and invasion of thyroid tumor cells [27]. In PC3 prostate cancer cells autocrine TGFβ1 signalling appears to be involved in hypoxia-mediated secretion of the vascular endothelial growth factor A (VEGFA). Darrington et al. suggested a working model to explain the connections between hypoxia, TGFβ1 and VEGFA signalling: PC3 prostate cancer cells express VEGFR-2 protein that enables an autocrine VEGFA signalling. The VEGFA secretion is induced by both hypoxia and TGFβ1 and results in increased migration of PC3 cells. Furthermore, hypoxia directly stimulates TGFβ1 expression, which constitutes an autocrine mechanism to enhance the effects of hypoxia on VEGFA expression [28]. The influence of VEGFA on tumor cell migration has also been investigated in melanoma cells. In 2005, Lacal et al. analyzed the migratory behavior of human melanoma cells that simultaneously
] (]]]]) ]]]–]]]
5
produce VEGFA and express VEGFRs. In these cells a VEGFAdriven autocrine loop is responsible for their increased invasiveness to the extracellular matrix (ECM) compared to melanoma cells not expressing either VEGFA or VEGFRs [29]. In addition to the VEGFR/VEGFA system, the EGFR signalling may contribute to the tumorigenic potential of human melanoma cells. EGFR expression or overexpression has been proven in many melanoma cells as well as the secretion of different growth factors that activate EGFR, such as TGFα, EGF, amphiregulin, betacellulin and HB-EGF [30–36]. Meierjohann et al. could show that the mutationally activated EGFR variant Xmrk, which is responsible for the development of highly malignant tumors in Xiphophorus fish, leads to a clear increase of pigment cell motility. EGF stimulation of Xmrk induces the formation of a receptor/Fyn/ focal adhesion kinase (FAK) complex, activation of FAK, and increased focal contact and actin cytoskeleton turnover [37]. Advanced studies indicated that Xmrk mediates the production of biologically active secreted EGFR ligands. These ligands are able to enhance the activation of already pre-dimerized receptors in an autocrine manner [38]. In the present study we supposed an autocrine activation of EGFR to be involved in the regulation of pancreatic cancer cell migration. We demonstrated that CFPAC1 and IMIM-PC2 human pancreatic cancer cells express high levels of both EGFR and ErbB2 and only low levels of ErbB3 (Fig. 1). Similar to our results Ioannou et al. reported in the past that seven pancreatic cancer cell lines are positive for EGFR and ErB2, negative for ErbB4 and express extremely low or undetectable levels of ErbB3 [39]. Besides, several studies revealed an expression or even an overexpression of EGF, EGFR or the combination of EGF and EGFR in pancreatic carcinomas [40–42]. Furthermore, Murphy et al. identified an EGFR dependent autocrine growth pathway in pancreatic cancer cells [21]: when pancreatic cancer cells were grown without change of medium, proliferation was greater than when either medium was replaced frequently or cells were grown in the presence of the EGF receptor tyrosine kinase inhibitor AG1478. As a consequence of this, the authors suggest that soluble endogenous EGF-like growth factors mediate an autocrine EGFR activation and thus stimulate the proliferation of pancreatic cancer cells [21]. In light of these findings we hypothesized that autocrine EGFR and/or ErbB2 activation might be the reason for the constitutive PLCγ activation in pancreatic cancer cells. According to this model, EGFR and/or ErbB2 should be already activated in the cells without any exogenous growth factor treatment. Therefore, we performed migratory experiments with AG1478, the ErbB2 inhibitor 4-(3-Phenoxyphenyl)-5-cyano-2H-1,2,3-triazole and a neutralizing anti-EGFR antibody. All three substances were able to significantly inhibit the spontaneous migratory activity of CFPAC1 and IMIM-PC2 cells (Figs. 2 and 3), indicating a constitutive autocrine activation of EGFR and ErbB2. As no direct ligand for ErbB2 has yet been discovered and ErbB2 plays a role in the potentiation of ErbB receptor signalling as the preferred heterodimerization partner of all other ErbB family members [43–46], we focused the following experiments on EGFR signalling. Treatment with exogenous EGF increased the migratory activity of both pancreatic cancer cell lines (Fig. 3), suggesting that autocrine activation via the endogenously produced EGF was under our cell culture condition not maximally stimulating cells. Because of this induction of the migratory activity by exogenous EGF and the constitutive activation of EGFR potentially caused by endogenous
Please cite this article as: A.-M. Stock, et al., Induction of pancreatic cancer cell migration by an autocrine epidermal growth factor receptor activation, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.04.022
6
324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383
E XP E RI ME N TAL CE L L R ES E ARC H
EGF, we used an EGF immunoassay to quantify the EGF levels in the cell culture supernatants. Over a period of four days we could measure a continuous increase of the EGF concentration in the cell culture supernatants of both cell lines (Fig. 4), providing the direct evidence that CFPAC1 and IMIM-PC2 cells release EGF. This EGF secretion is a likely explanation for the constitutive EGFR activation, which leads to the previously described constitutive PLCγ phosphorylation [10]. As a consequence, the pancreatic cancer cells exhibit a very high spontaneous migratory activity that enables a high frequency of early invasion and metastasis development. Additional EGFR ligands different from EGF might contribute to this autocrine effect, too. However, these ligands have not been studied herein. To date, several antibodies and tyrosine kinase inhibitors targeting EGFR or ErbB2 have been approved for treatment of different human cancers: the anti-EGFR monoclonal antibody cetuximab for metastatic colorectal cancer, head and neck cancer; the anti-EGFR monoclonal antibody panitumumab for metastatic colorectal cancer; the EGFR tyrosine kinase inhibitors gefitinib and erlotinib for non-small cell lung cancer; the anti-ErbB2 monoclonal antibody trastuzumab for metastatic stomach cancers and breast cancer and the anti-ErbB2 monoclonal antibody pertuzumab as well as the dual EGFR/ErbB2 tyrosine kinase inhibitor lapatinib for breast cancer [47–50]. For the treatment of patients with pancreatic cancer only the EGFR tyrosine kinase inhibitor erlotinib has gained the FDA approval in combination with the chemotherapeutic drug gemcitabine [51]. EGFR and ErbB2 are not the only receptor tyrosine kinases, which might be responsible for the high spontaneous migration of pancreatic cancer cells via induction of PLCγ phosphorylation. As introduced before, the PLCγ is also a substrate for PDGF receptor tyrosine kinases [22,23]. Overexpression of PDGF ligands and receptors has been reported for many solid tumors including glioblastoma, meningioma, melanoma, ovarian cancer, prostate cancer, lung cancer, gastric carcinoma and pancreatic cancer [52]. Kawaguchi et al. studied the involvement of PDGF signalling in AsPC1 and BxPC3 pancreatic cancer cell migration and hypothesized that the homodimer PDGF-BB strongly induces cell migration via the Akt pathway [53]. In the present study flow cytometric analyses revealed a low expression of PDGFRα and no detectable amounts of PDGFRβ in IMIM-PC2 and CFPAC1 pancreatic cancer cells (Fig. 1). In subsequent migratory experiments a neutralizing anti-PDGFRα antibody significantly reduced the migratory activity of IMIM-PC2 cells. In CFPAC1 cells the antibody did not cause any significant effects (Fig. 5). Therefore, we assume that a PDGFRα activation contributes to the autocrine stimulation in IMIM-PC2 cells, but not in CFPAC1 cells. Further growth factor receptors or cytokine receptors might be involved, which were not investigated in this study.
Conclusion We suggest that autocrine EGFR activation by secreted EGF induces constitutive PLCγ phosphorylation and finally results in a high spontaneous migratory activity of pancreatic cancer cells. Besides this, the migratory experiments with the neutralizing PDGFRα antibody show the individuality of each cell line: In IMIM-PC2 cells PDGFRα seems to be involved, too, in CFPAC1 cells
] (]]]]) ]]]–]]]
not. Thus, our results and the literature discussed herein provide the evidence for the importance of growth factors and their receptors as targets for the development of anti-metastatic drugs, which have to be individually adjusted to the patients.
Acknowledgments This work was supported by the Fritz Bender Foundation (Munich, Q2 Germany).
references [1] T. Schnelldorfer, A.L. Ware, M.G. Sarr, T.C. Smyrk, L. Zhang, R. Qin, R.E. Gullerud, J.H. Donohue, D.M. Nagorney, M.B. Farnell, Longterm survival after pancreatoduodenectomy for pancreatic adenocarcinoma: is cure possible?, Ann. Surg. 247 (2008) 456–462. [2] J. Guo, W. Lou, Y. Ji, S. Zhang, Effect of CCR7, CXCR4 and VEGF-C on the lymph node metastasis of human pancreatic ductal adenocarcinoma, Oncol. Lett. 5 (2013) 1572–1578. [3] F. Entschladen, T.L.t. Drell, K. Lang, J. Joseph, K.S. Zänker, Tumourcell migration, invasion, and metastasis: navigation by neurotransmitters, Lancet Oncol. 5 (2004) 254–258. [4] D. Kedrin, J. van Rheenen, L. Hernandez, J. Condeelis, J.E. Segall, Cell motility and cytoskeletal regulation in invasion and metastasis, J. Mammary Gland Biol. Neoplasia 12 (2007) 143–152. [5] F. Balkwill, A. Mantovani, Inflammation and cancer: back to Virchow?, Lancet 357 (2001) 539–545. [6] F. Entschladen, K.S. Zänker, D.G. Powe, Heterotrimeric G protein signaling in cancer cells with regard to metastasis formation, Cell Cycle 10 (2011) 1086–1091. [7] F. Entschladen, T.L.t. Drell, K. Lang, J. Joseph, K.S. Zänker, Neurotransmitters and chemokines regulate tumor cell migration: potential for a new pharmacological approach to inhibit invasion and metastasis development, Curr. Pharm. Des. 11 (2005) 403–411. [8] P. Bastian, A. Balcarek, C. Altanis, C. Strell, B. Niggemann, K.S. Zaenker, F. Entschladen, The inhibitory effect of norepinephrine on the migration of ES-2 ovarian carcinoma cells involves a Rap1-dependent pathway, Cancer Lett. 274 (2009) 218–224. [9] A.M. Stock, G. Troost, B. Niggemann, K.S. Zanker, F. Entschladen, Targets for anti-metastatic drug development, Curr. Pharm. Des. (2013). [10] A.M. Stock, D.G. Powe, S.A. Hahn, G. Troost, B. Niggemann, K.S. Zanker, F. Entschladen, Norepinephrine inhibits the migratory activity of pancreatic cancer cells, Exp. Cell Res. 319 (2013) 1744–1758. [11] B. Niggemann, T.L.t. Drell, J. Joseph, C. Weidt, K. Lang, K.S. Zaenker, F. Entschladen, Tumor cell locomotion: differential dynamics of spontaneous and induced migration in a 3D collagen matrix, Exp. Cell Res. 298 (2004) 178–187. [12] C. Strell, B. Niggemann, M.J. Voss, D.G. Powe, K.S. Zanker, F. Entschladen, Norepinephrine promotes the beta1-integrinmediated adhesion of MDA-MB-231 cells to vascular endothelium by the induction of a GROalpha release, Mol. Cancer Res. 10 (2012) 197–207. [13] D.L. Cadena, G.N. Gill, Receptor tyrosine kinases, FASEB J. 6 (1992) 2332–2337. [14] G. Alonso, M. Koegl, N. Mazurenko, S.A. Courtneidge, Sequence requirements for binding of Src family tyrosine kinases to activated growth factor receptors, J. Biol. Chem. 270 (1995) 9840–9848. [15] N.R. Lemoine, C.M. Hughes, C.M. Barton, R. Poulsom, R.E. Jeffery, G. Kloppel, P.A. Hall, W.J. Gullick, The epidermal growth factor receptor in human pancreatic cancer, J. Pathol. 166 (1992) 7–12.
Please cite this article as: A.-M. Stock, et al., Induction of pancreatic cancer cell migration by an autocrine epidermal growth factor receptor activation, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.04.022
E X PE R IM EN TA L C ELL R E S EA RC H
384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443
[16] K. Kimura, T. Sawada, M. Komatsu, M. Inoue, K. Muguruma, T. Nishihara, Y. Yamashita, N. Yamada, M. Ohira, K. Hirakawa, Antitumor effect of trastuzumab for pancreatic cancer with high HER-2 expression and enhancement of effect by combined therapy with gemcitabine, Clin. Cancer Res. 12 (2006) 4925–4932. [17] M. Ebert, M. Yokoyama, H. Friess, M.S. Kobrin, M.W. Buchler, M. Korc, Induction of platelet-derived growth factor A and B chains and over-expression of their receptors in human pancreatic cancer, Int. J. Cancer 62 (1995) 529–535. [18] Y. Yarden, M.X. Sliwkowski, Untangling the ErbB signalling network, Nat. Rev. Mol. Cell Biol. 2 (2001) 127–137. [19] A. Citri, Y. Yarden, EGF-ERBB signalling: towards the systems level, Nat. Rev. Mol. Cell Biol. 7 (2006) 505–516. [20] P. Seshacharyulu, M.P. Ponnusamy, D. Haridas, M. Jain, A.K. Ganti, S.K. Batra, Targeting the EGFR signaling pathway in cancer therapy, Expert Opin. Ther. Targets 16 (2012) 15–31. [21] L.O. Murphy, M.W. Cluck, S. Lovas, F. Otvos, R.F. Murphy, A.V. Schally, J. Permert, J. Larsson, J.A. Knezetic, T.E. Adrian, Pancreatic cancer cells require an EGF receptor-mediated autocrine pathway for proliferation in serum-free conditions, Br. J. Cancer 84 (2001) 926–935. [22] H.K. Kim, J.W. Kim, A. Zilberstein, B. Margolis, J.G. Kim, J. Schlessinger, S.G. Rhee, PDGF stimulation of inositol phospholipid hydrolysis requires PLC-gamma 1 phosphorylation on tyrosine residues 783 and 1254, Cell 65 (1991) 435–441. [23] J. Meisenhelder, P.G. Suh, S.G. Rhee, T. Hunter, Phospholipase C-gamma is a substrate for the PDGF and EGF receptor proteintyrosine kinases in vivo and in vitro, Cell 57 (1989) 1109–1122. [24] K. Masur, K. Lang, B. Niggemann, K.S. Zänker, F. Entschladen, PKC High, alpha and low E-cadherin expression contribute to high migratory activity of colon carcinoma cells, Mol. Biol. Cell 12 (2001) 1973–1982. [25] D. Hanahan, R.A. Weinberg, Hallmarks of cancer: the next generation, Cell 144 (2011) 646–674. [26] R. Hasina, K. Matsumoto, N. Matsumoto-Taniura, I. Kato, M. Sakuda, T. Nakamura, Autocrine and paracrine motility factors and their involvement in invasiveness in a human oral carcinoma cell line, Br. J. Cancer 80 (1999) 1708–1717. [27] G. Riesco-Eizaguirre, I. Rodriguez, A. De la Vieja, E. Costamagna, N. Carrasco, M. Nistal, P. Santisteban, The BRAFV600E oncogene induces transforming growth factor beta secretion leading to sodium iodide symporter repression and increased malignancy in thyroid cancer, Cancer Res. 69 (2009) 8317–8325. [28] E. Darrington, M. Zhong, B.H. Vo, S.A. Khan, Vascular endothelial growth factor A, secreted in response to transforming growth factor-beta1 under hypoxic conditions, induces autocrine effects on migration of prostate cancer cells, Asian J. Androl. 14 (2012) 745–751. [29] P.M. Lacal, F. Ruffini, E. Pagani, S. D’Atri, An autocrine loop directed by the vascular endothelial growth factor promotes invasiveness of human melanoma cells, Int. J. Oncol. 27 (2005) 1625–1632. [30] K. Krasagakis, C. Garbe, C.C. Zouboulis, C.E. Orfanos, Growth control of melanoma cells and melanocytes by cytokines, Recent Results Cancer Res. 139 (1995) 169–182. [31] B. Singh, M. Schneider, P. Knyazev, A. Ullrich, UV-induced EGFR signal transactivation is dependent on proligand shedding by activated metalloproteases in skin cancer cell lines, Int. J. Cancer 124 (2009) 531–539. [32] N. Bardeesy, M. Kim, J. Xu, R.S. Kim, Q. Shen, M.W. Bosenberg, W.H. Wong, L. Chin, Role of epidermal growth factor receptor signaling in RAS-driven melanoma, Mol. Cell Biol. 25 (2005) 4176–4188. [33] L.A. Akslen, H. Puntervoll, I.M. Bachmann, O. Straume, E. Vuhahula, R. Kumar, A. Molven, Mutation analysis of the EGFR-NRAS-BRAF pathway in melanomas from black Africans
[34]
[35]
[36]
[37]
[38]
[39]
[40]
[41]
[42]
[43]
[44]
[45]
[46]
[47]
[48]
[49]
[50]
] (]]]]) ]]]–]]]
7
and other subgroups of cutaneous melanoma, Melanoma Res. 18 (2008) 29–35. P.E. de Wit, S. Moretti, P.G. Koenders, M.A. Weterman, G.N. van Muijen, B. Gianotti, D.J. Ruiter, Increasing epidermal growth factor receptor expression in human melanocytic tumor progression, J. Invest. Dermatol. 99 (1992) 168–173. S. Mattei, M.P. Colombo, C. Melani, A. Silvani, G. Parmiani, M. Herlyn, Expression of cytokine/growth factors and their receptors in human melanoma and melanocytes, Int. J. Cancer 56 (1994) 853–857. L.E. Sparrow, P.J. Heenan, Differential expression of epidermal growth factor receptor in melanocytic tumours demonstrated by immunohistochemistry and mRNA in situ hybridization, Australas. J. Dermatol. 40 (1999) 19–24. S. Meierjohann, E. Wende, A. Kraiss, C. Wellbrock, M. Schartl, The oncogenic epidermal growth factor receptor variant Xiphophorus melanoma receptor kinase induces motility in melanocytes by modulation of focal adhesions, Cancer Res. 66 (2006) 3145–3152. J.A. Laisney, T.D. Mueller, M. Schartl, S. Meierjohann, Hyperactivation of constitutively dimerized oncogenic EGF receptors by autocrine loops, Oncogene 32 (2013) 2403–2411. N. Ioannou, A.G. Dalgleish, A.M. Seddon, D. Mackintosh, U. Guertler, F. Solca, H. Modjtahedi, Anti-tumour activity of afatinib, an irreversible ErbB family blocker, in human pancreatic tumour cells, Br. J. Cancer 105 (2011) 1554–1562. K. Uegaki, Y. Nio, Y. Inoue, Y. Minari, Y. Sato, M.M. Song, M. Dong, K. Tamura, Clinicopathological significance of epidermal growth factor and its receptor in human pancreatic cancer, Anticancer Res. 17 (1997) 3841–3847. M. Bloomston, A. Bhardwaj, E.C. Ellison, W.L. Frankel, Epidermal growth factor receptor expression in pancreatic carcinoma using tissue microarray technique, Dig. Surg. 23 (2006) 74–79. K. Tobita, H. Kijima, S. Dowaki, H. Kashiwagi, Y. Ohtani, Y. Oida, H. Yamazaki, M. Nakamura, Y. Ueyama, M. Tanaka, S. Inokuchi, H. Makuuchi, Epidermal growth factor receptor expression in human pancreatic cancer: Significance for liver metastasis, Int. J. Mol. Med. 11 (2003) 305–309. E. Tzahar, H. Waterman, X. Chen, G. Levkowitz, D. Karunagaran, S. Lavi, B.J. Ratzkin, Y. Yarden, A hierarchical network of interreceptor interactions determines signal transduction by Neu differentiation factor/neuregulin and epidermal growth factor, Mol. Cell. Biol. 16 (1996) 5276–5287. D. Graus-Porta, R.R. Beerli, J.M. Daly, N.E. Hynes, ErbB-2, the preferred heterodimerization partner of all ErbB receptors, is a mediator of lateral signaling, EMBO J. 16 (1997) 1647–1655. R.R. Beerli, D. Graus-Porta, K. Woods-Cook, X. Chen, Y. Yarden, N.E. Hynes, Neu differentiation factor activation of ErbB-3 and ErbB-4 is cell specific and displays a differential requirement for ErbB-2, Mol. Cell. Biol. 15 (1995) 6496–6505. D. Graus-Porta, R.R. Beerli, N.E. Hynes, Single-chain antibodymediated intracellular retention of ErbB-2 impairs Neu differentiation factor and epidermal growth factor signaling, Mol. Cell. Biol. 15 (1995) 1182–1191. N. Ioannou, A.M. Seddon, A. Dalgleish, D. Mackintosh, H. Modjtahedi, Expression pattern and targeting of HER family members and IGF-IR in pancreatic cancer, Front. Biosci. 17 (2012) 2698– 2724. C.M. Rocha-Lima, L.E. Raez, Erlotinib (tarceva) for the Treatment of Non-small-cell Lung Cancer and Pancreatic Cancer, P T 34 (2009) 554–564. K.S. Zaenker, F. Entschladen, Paving roads for new drugs in oncology, Recent Patents Anticancer Drug Discovery 4 (2009) 137–145. N. Ioannou, A.M. Seddon, A. Dalgleish, D. Mackintosh, H. Modjtahedi, Treatment with a combination of the ErbB (HER) family blocker afatinib and the IGF-IR inhibitor, NVP-AEW541 induces
Please cite this article as: A.-M. Stock, et al., Induction of pancreatic cancer cell migration by an autocrine epidermal growth factor receptor activation, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.04.022
8
444 445 446 447 448 449 450 451
E XP E RI ME N TAL CE L L R ES E ARC H
synergistic growth inhibition of human pancreatic cancer cells, BMC Cancer 13 (2013) 41. [51] R.K. Kelley, A.H. Ko, Erlotinib in the treatment of advanced pancreatic cancer, Biologics 2 (2008) 83–95. [52] A. Ostman, C.H. Heldin, Involvement of platelet-derived growth factor in disease: development of specific antagonists, Adv. Cancer Res. 80 (2001) 1–38.
] (]]]]) ]]]–]]]
[53] J. Kawaguchi, S. Adachi, I. Yasuda, T. Yamauchi, T. Yoshioka, M. Itani, O. Kozawa, H. Moriwaki, UVC irradiation suppresses platelet-derived growth factor-BB-induced migration in human pancreatic cancer cells, Oncol. Rep. 27 (2012) 935–939.
Please cite this article as: A.-M. Stock, et al., Induction of pancreatic cancer cell migration by an autocrine epidermal growth factor receptor activation, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.04.022
24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41Q1 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83
] (]]]]) ]]]–]]]
Available online at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/yexcr
Research Article
Induction of pancreatic cancer cell migration by an autocrine epidermal growth factor receptor activation Anna-Maria Stocka, Stephan A Hahnb, Gabriele Troosta, Bernd Niggemanna, Kurt S Zänkera, Frank Entschladena,n a
Institute of Immunology and Experimental Oncology, Centre for Biomedical Education and Research (ZBAF), Witten/Herdecke University, 58448 Witten, Germany b Department of Molecular Gastroenterological Oncology, Centre of Clinical Research, Ruhr-University of Bochum, 44780 Bochum, Germany
article information
abstract
Article Chronology:
Pancreatic cancer is characterized by aggressive local invasion and early metastasis formation. Active
Received 17 February 2014
migration of the pancreatic cancer cells is essential for these processes. We have shown previously that
Received in revised form
the pancreatic cancer cells lines CFPAC1 and IMIM-PC2 show high migratory activity, and we have
24 April 2014
investigated herein the reason for this observation. Cell migration was assessed using a three-
Accepted 28 April 2014
dimensional, collagen-based assay and computer-assisted cell tracking. The expression of receptor tyrosine kinases was determined by flow-cytometry and cytokine release was measured by an
Keywords: Pancreatic cancer Metastasis Cell migration Receptor tyrosine kinases Epidermal growth factor
enzyme-linked immunoassay. Receptor function was blocked by antibodies or pharmacological enzyme inhibitors. Both cells lines express the epidermal growth factor receptor (EGFR) as well as its familymember ErbB2 and the platelet-derived growth factor receptor (PDGFR)α, whereas only weak expression was detected for ErbB3 and no expression of PDGFRβ. Pharmacological inhibition of the EGFR or ErbB2 significantly reduced the migratory activity in both cell lines, as did an anti-EGFR antibody. Interestingly, combination of the latter with an anti-PDGFR antibody led to an even more pronounced reduction. Both cell lines release detectable amounts of EGF. Thus, the high migratory activity of the investigated pancreatic cancer cell lines is due to autocrine EGFR activation and possibly of other receptor tyrosine kinases. & 2014 Published by Elsevier Inc.
Introduction The difficult diagnosis of pancreatic cancer at a localized resectable stage makes the prognosis of patients with this disease extremely poor. Another reason of the poor prognosis is the early metastasis formation of the tumor to the regional lymph nodes and liver [1,2].
An essential prerequisite for metastasis formation is the active migration of malignant cells from the primary tumor via lymphatic or blood vessel routes. This complex process of cancer cell migration is regulated by many different signalling pathways as well as substances of various classes and origins [3]. The most important migrational regulators can be divided into two major groups: On the
Abbreviations: cAMP, cyclic AMP; EGF(R), epidermal growth factor (receptor); EMT, epithelial to mesenchymal transition; FITC, fluorescein isothiocyanate; FAK, focal adhesion kinase; GPCR, G protein-coupled receptor; mAb, monoclonal antibody; PI3, phosphatidyl-inositide 3; PLC, phospholipase C; HB-EGF, heparin-binding EGF-like growth factor; PDGF(R), platelet-derived growth factor (receptor); PKA/C, protein kinase A/C; TGF, transforming growth factor; VEGF(R), vascular endothelial growth factor(R) n
Corresponding author. Fax: þ49 2302 926 158. E-mail address: [email protected] (F. Entschladen).
http://dx.doi.org/10.1016/j.yexcr.2014.04.022 0014-4827/& 2014 Published by Elsevier Inc.
Please cite this article as: A.-M. Stock, et al., Induction of pancreatic cancer cell migration by an autocrine epidermal growth factor receptor activation, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.04.022
2
84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143
E XP E RI ME N TAL CE L L R ES E ARC H
one hand there are cytokines and growth factors, which regulate receptor kinases and related receptors with associated kinases [4,5]. The second group includes chemokines and neurotransmitters, which are able to activate members of the huge family of G protein coupled receptors (GPCRs) [6,7]. One example of these GPCR ligands is the stress catecholamine hormone norepinephrine. In previous in vitro cell migration studies we have observed that norepinephrine induces diverse effects on the migratory activity of different human tumor cells: some cell lines increase their migratory activity in response to norepinephrine, some do not show any significant norepinephrineinduced effects and others reduce their migratory activity after norepinephrine treatment [8–12]. The pancreatic cancer cell lines CFPAC1 and IMIM-PC2 are part of the last named group. The inhibitory effect of norepinephrine on the migration of these cells appears to be due to an imbalance of two cell signaling pathways. The protein kinase C/phospholipase C (PKC/ PLC) pathway is already activated in the absence of norepinephrine, leading to an activation of the motor protein non-muscle myosin II and thus to a high spontaneous migratory activity. Therefore, norepinephrine does not cause further activation of this PLC/PKC pathway, but instead activates the second cyclic AMP/protein kinase A (cAMP/PKA)-dependent pathway and finally leads to an inhibitory effect on pancreatic cancer cell migration [10]. The signalling pathway or signalling event, which is responsible for the constitutive PLCγ activation in pancreatic cancer cells, is not clear. We hypothesize that an autocrine activation of receptor tyrosine kinases might be the reason for the constitutive PLCγ activation. Ligand binding induces dimerization and autophosphorylation of receptor tyrosine kinases and results in phosphorylation of various SH2 domain-containing signal transduction molecules such as PLCγ, PI3-kinase, GAP and cSrc [13,14]. Examples of these receptor tyrosine kinases are the platelet-derived growth factor receptors α and β (PDGFRα and PDGFRβ) and the four members of the ErbB receptor family: the epidermal growth factor receptor (EGFR/ErbB1), HER2/neu (ErbB2), HER3 (ErbB3) and HER4 (ErbB4). The expression of the PDGF receptors and at least one of the members of the EGFR family has been proven in a large proportion of pancreatic cancers [15–17]. The activation of the different ErbB receptors can be induced by the following ligands: EGF, transforming growth factor alpha (TGF-α), amphiregulin, betacellulin, heparin-binding EGF-like growth factor (HB-EGF), epiregulin, epigen and neuregulins 1–6 [18,19]. Among these ligands, TGF-α, amphiregulin, betacellulin, epiregulin and EGF are specific activators of EGFR [20]. A study of Murphy et al. revealed that autocrine EGFR activation stimulates the proliferation of these cells [21]. Furthermore, different studies indicated that EGF and PDGF initiate the association of their respective receptors with PLCγ and induce the PLCγ phosphorylation [22,23]. In the context of these findings the present study focuses on the role of EGFR, ErbB2 and PDGFR in pancreatic cancer cell migration and provides EGF as an important autocrine factor for the regulation of the migratory activity of these cells.
Materials and methods Cell culture The pancreatic cancer cell line CFPAC1 was purchased from American Type Culture Collection (Manassas, VA, USA) and the pancreatic cancer cell line IMIM-PC2 was a gift from Francisco
] (]]]]) ]]]–]]]
X Real (Spanish National Cancer Research Centre, Madrid, Spain). The cells were grown in Dulbecco's Modified Eagle Medium (Sigma-Aldrich, Steinheim, Germany), supplemented with 10% fetal bovine serum (Biochrom AG, Berlin, Germany), 2% penicillin/streptomycin (Sigma-Aldrich), 1 mM sodium pyruvate (Biochrom) and 2 mM L-glutamine (PAA Laboratories GmbH, Linz, Austria) at 37 1C in a humidified atmosphere of 5% CO2. For the pancreatic cancer cell line CFPAC-1 reference STR data are available The GenomeLab Human STR Primer Set (Beckman Coulter, Krefeld, Germany) was used and analyzed on a CEQ8800 sequencer (Beckman Coulter) according to the manufacturer's protocol to control for the cell line identity. STR data were submitted to on-line verification tool of DSMZ (German Collection of Microorganisms and Cell Lines) to confirm identity (http://old. dsmz.de/human_and_animal_cell_lines/main.php?contentle ft_id=101). For IMIM-PC2, there is no reference available.
Cell migration assay The migratory activity of the two cell lines was analyzed as described in detail previously [24]. In short, 5 104 cells were mixed with carbonate-buffered collagen solution (Advanced BioMatrix, San Diego, CA, USA) containing minimal essential medium (Sigma-Aldrich) as well as one or two of the following substances: the EGFR kinase inhibitor AG1478 (300 nM, Merck KGaA, Darmstadt, Germany), EGF (100 ng/ml, Sigma-Aldrich), the ErbB2 inhibitor 4-(3-Phenoxyphenyl)-5-cyano-2H-1,2,3-triazole (10 mM, Merck KGaA), a neutralizing anti-EGFR antibody (10 mg/ml, Merck KGaA) and a neutralizing anti-PDGFRα antibody (10 mg/ml, Abcam, Cambridge, UK). After polymerization of the collagen, the migration of the cells was recorded by time-lapse video microscopy for 15 h. Subsequently two-dimensional projections of the paths of 30 randomly selected cells were digitized in 15 min intervals. The migratory activity was calculated for each step as the portion of cells, which was locomotory active. The average of the activity was calculated for all steps of the entire observation period. The graphs show the mean values and standard deviations of the migratory activity of at least three independent experiments. In minimum 90 cells were analyzed per sample. Statistically significant changes (po0.05) were calculated using Student's t-test (two-tailed and unpaired).
Flow cytometry A FacsCalibur flow cytometer (Becton Dickinson GmbH, Heidelberg, Germany) was used to investigate the expression of EGFR, ErbB2, ErbB3, PDGFRα and β in pancreatic cancer cells. 1 105 cells were fixed with 1% paraformaldehyde (SigmaAldrich), washed with 0.5% Triton X-100 (Sigma-Aldrich) and incubated with one of the following antibodies (Ab): anti-EGFR (Ab-1) mouse mAb (528) IgG2a (200 ng/ml, Merck KGaA), isotypic control IgG2a (1:250 dilution, Beckman Coulter, Marseille, France), anti-c-ErbB2/c-Neu (Ab-2) mouse mAb 9G6 (200 ng/ml, Merck KGaA), ErbB-3 (298) mouse monoclonal IgG1 (200 ng/ml, Santa Cruz Biotechnology, Heidelberg, Germany), isotypic control IgG1 (400 ng/ml, Beckman Coulter), PDGFRα (D13C6) XP™ rabbit mAb (1:50 dilution, New England Biolabs GmbH, Frankfurt), PDGFRβ (C82A3) rabbit mAb (1:50 dilution, New England Biolabs GmbH) or normal rabbit IgG (1:50 dilution, Santa Cruz Biotechnology). After 30 min incubation one of the following secondary
Please cite this article as: A.-M. Stock, et al., Induction of pancreatic cancer cell migration by an autocrine epidermal growth factor receptor activation, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.04.022
E X PE R IM EN TA L C ELL R E S EA RC H
In contrast to this high EGFR and ErbB2 expression, a faint expression of ErbB3 and PDGFRα was observed in both cell lines. The ErbB3 mean fluorescence intensity was 5.91 for the CFPAC1 and 2.22 for the IMIM-PC2 cells and the PDGFRα mean fluorescence intensity was 5.13 for the CFPAC1 and 7.62 for the IMIM-PC2 cells. Both cell lines contained no detectable amounts of PDGFRβ.
CFPAC1
Measurement of EGF The Quantikines ELISA Human EGF Immunoassay (R&D Systems Europe, Abingdon, UK) was used for the quantitative determination of human EGF concentrations in cell culture supernatants. The sample preparation and the measurement of the EGF concentrations were performed according to the manufacturer’s protocol. Cells (1 106 cells/ml) were seeded into 24-well-plates and incubated at 37 1C in a humidified atmosphere of 5% CO2. Samples were harvested every 24 h by centrifugation and stored at 20 1C. Every experiment was repeated three times. All standards and samples of one experiment were assayed in triplicate.
Results In previous studies we could show that the pancreatic cancer cell lines CFPAC1 and IMIM-PC2 developed a high spontaneous migratory activity after incorporation within a three-dimensional collagen matrix. Additionally, our results demonstrated that non-muscle myosin II seems to be constitutively activated via the PKC/PLC pathway in these cells [10]. According to these findings and the link between the PLCγ phosphorylation, EGFR and PDGFR [22,23], we analyzed the role of EGFR and PDGFR in the regulatory network of pancreatic cancer cell migration. Therefore, we investigated the expression of EGFR, ErbB2, ErbB3, PDGFRα and PDGFRβ on the cell surface of CFPAC1 and IMIM-PC2 cells by flow cytometric analysis (Fig. 1). In both cell lines the highest mean fluorescence intensity was detectable in the case of the EGF receptor: 24.18 for the CFPAC1 cells and 24.62 for the IMIM-PC2 cells. The mean fluorescence intensities of 23.62 (CFPAC1) and 11.87 (IMIM-PC2) indicated that the ErbB2 expression was slightly lower than the EGFR expression.
100
Locomoting cells (%)
antibodies was added: R-phycoerythrin-conjugated AffiniPure F (ab')2 fragment goat anti-mouse IgG (1:250 dilution, Dianova GmbH, Hamburg, Germany) or goat anti-rabbit IgG-FITC (8 mg/ml, Santa Cruz Biotechnology). All incubation steps were performed at room temperature. Additionally, the viability of the cells was assessed by propidium iodide staining and flow cytometry in order to exclude that a reduction of migratory activity might be due to cell death.
90 80 70 60 50 40 30 20 10 0
IMIM-PC2
Control
AG1478
ErbB2 Inhibitor
Control
AG1478
ErbB2 Inhibitor
100
Locomoting cells (%)
144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203
3
] (]]]]) ]]]–]]]
90 80 70 60 50 40 30 20 10 0
Fig. 2 – Migratory activity of pancreatic carcinoma cells in response to the EGFR kinase inhibitor AG1478 and the ErbB2 inhibitor 4-(3-Phenoxyphenyl)-5-cyano-2H-1,2,3-triazole. AG1478 was applied at a concentration of 300 nM and the ErbB2 inhibitor was applied at a concentration of 10 lM. The graphs show mean values and standard deviations of at least three independent experiments. Asterisks mark statistically significant changes (po0.05).
Fig. 1 – Expression of EGFR, ErbB2, ErbB3, PDGFRα and PDGFRβ on pancreatic cancer cells. Gray fields¼isotypic control antibody (ISO); black line ¼expression of EGFR, ErbB2 and ErbB3; MFI ¼mean fluorescence intensity. Please cite this article as: A.-M. Stock, et al., Induction of pancreatic cancer cell migration by an autocrine epidermal growth factor receptor activation, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.04.022
4
CFPAC1
100
Locomoting cells (%)
] (]]]]) ]]]–]]]
12
90 80
*
70
*
*
60
EGF (pg/ml)
CFPAC1
50 40 30 20 10
10 8 6 4
0
Control
EGF
EGFR EGFR antibody antibody & EGF
2 0
100
*
90
*
*
80 70
day 1
day 2
day 3
day 4
day 1
day 2
day 3
day 4
IMIM-PC2 12
60 50 40
EGF (pg/ml)
IMIM-PC2 Locomoting cells (%)
204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263
E XP E RI ME N TAL CE L L R ES E ARC H
30 20 10 0
Control
EGF
EGFR EGFR antibody antibody & EGF
Fig. 3 – Migratory activity of pancreatic carcinoma cells in response to EGF and an EGFR neutralizing antibody. EGF was applied at a concentration of 100 ng/ml and the EGFR neutralizing antibody was applied at a concentration of 10 lg/ml. The graphs show mean values and standard deviations of at least three independent experiments. Asterisks mark statistically significant changes (po0.05).
The flow cytometric analyses were followed by migratory experiments with AG1478 (300 nM; Fig. 2). This EGFR kinase inhibitor significantly decreased the spontaneous migratory activity of CFPAC1 cells from 47.4%71.6% to 33.8%74.4% (p¼ 0.004) and the activity of IMIM-PC2 cells from 48.4%76.8% to 29.6%75.2% (po0.001). On the basis of the high ErbB2 expression we also evaluated the effects of ErbB2 inhibition on pancreatic cancer cell migration (Fig. 2). The ErbB2 inhibitor 4-(3-Phenoxyphenyl)-5-cyano-2H-1,2,3-triazole (10 mM) significantly reduced the migratory activity of CFPAC1 cells from 47.4%71.6% to 28.3%74.0% (po0.001) and the migratory activity of IMIM-PC2 cells from 48.4%76.8% to 32.3%74.9% (p¼ 0.003). Furthermore, we found that treatment with 100 ng/ml of the EGFR ligand EGF augmented the spontaneous migratory activity of the CFPAC1 cells significantly (po0.001) from 40.3%72.3% to 51.9% 71.7% and the migratory activity of the IMIM-PC2 cells from 49.4% 71.8% to 64.2%710.0% (p¼0.027; Fig. 3). A concentration of 10 mg/ml of a neutralizing anti-EGFR antibody was able to inhibit this EGF-induced increased migratory activity. CFPAC1 cells showed a significant decrease (p¼ 0.002) of locomoting cells from 51.9%71.7% to 34.2%76.0% and IMIM-PC2 cells changed from 64.2%710.0% to 36.1%76.8% locomoting cells (p¼0.009). In addition to the decrease of the EGF-induced migratory activity, the neutralizing anti-EGFR antibody caused a significant reduction of the spontaneous migratory activity. In the case of the CFPAC1 cells the migratory activity diminished from 40.3%72.3% to 31.6%75.4% (p¼0.032) and in the IMIM-PC2 cells from 49.4%71.8% to 26.4%76.0% (po0.001). Due to the induction of the migratory activity by EGF, we used the Quantikines ELISA Human EGF Immunoassay to analyze the EGF levels in the cell culture supernatants over a period of four days (Fig. 4). The EGF concentration in the supernatant of the
10 8 6 4 2 0
Fig. 4 – Quantitative determination of human EGF concentrations in cell culture supernatants. The graphs show the mean values and the standard deviations of the EGF concentrations in cell culture supernatants after one, two, three and four days of three independent experiments. CFPAC1 cells increased from 0.9 pg/ml70.6 pg/ml after 24 hours to 8.1 pg/ml 71.9 pg/ml after four days and in the supernatant of the IMIM-PC2 cells from 0.8 pg/ml70.2 pg/ml to 12.0 pg/ml70.6 pg/ml. The measured amounts of secreted EGF have not been adjusted to the increase of the cell number during the incubation period. However, the cell growth has been analyzed previously [10]. The increase of secreted EGF by far exceeds the growth of the cells within the observation period of four days. Thus, there is an accumulation of EGF in the culture supernatant over time. According to the link between PLCγ phosphorylation, EGFR and PDGFR introduced before, we used a neutralizing PDGFRα antibody to investigate the influence of PDGFRα inhibition on the locomotory activity of pancreatic cancer cells (Fig. 5). In CFPAC1 cells this antibody did not lead to a significant effect on the migratory activity, whereas the percentage of locomoting IMIMPC2 cells significantly decreased from 30.9%75.4% to 21.1%72.3% (p¼ 0.020). A combination of the neutralizing PDGFRα and the neutralizing EGFR antibody reduced the migratory activity of CFPAC1 cells significantly from 26.2%74.8% to 18.7%73.8% (p¼ 0.049) and in IMIM-PC2 cells from 30.9%75.4% to 16.9% 72.2% (p¼ 0.013). Therefore, it seems likely that not only EGF but further growth factor receptors or cytokine receptors such as the PDGFR are involved in this autocrine stimulation of migration.
Discussion The development of autocrine loops makes tumor cells independent of exogenous growth factors, which is regarded as a hallmark
Please cite this article as: A.-M. Stock, et al., Induction of pancreatic cancer cell migration by an autocrine epidermal growth factor receptor activation, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.04.022
E X PE R IM EN TA L C ELL R E S EA RC H
CFPAC1 Locomoting cells (%)
100 90 80 70 60 50 40 30 20 10 0
IMIM-PC2
Control
PDGFR PDGFR& antibody EGFR antibodies
Control
PDGFR PDGFR& antibody EGFR antibodies
100
Locomoting cells (%)
264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323
90 80 70 60 50 40 30 20 10 0
Fig. 5 – Migratory activity of pancreatic carcinoma cells in response to a PDGFRα neutralizing antibody and an EGFR neutralizing antibody. Both antibodies were applied at a concentration of 10 lg/ml. The graphs show mean values and standard deviations of at least three independent experiments. Asterisks mark statistically significant changes (po0.05).
of cancer [25]. Autocrine induction of cancer cell migration by different growth factors has been described for various types of tumor cells. In 1999, the transforming growth factors α and β1 (TGFα and TGFβ1) were identified as autocrine acting motility factors in oral squamous carcinoma cells, which stimulate the autonomous migration of these cells [26]. Another autocrine TGFβ loop was observed in papillary thyroid carcinoma cells. In this case the oncogene BRAF induces TGFβ secretion as a part of a sodium/iodide symporter (NIS) repression mechanism. The secreted TGFβ leads to an increased epithelial to mesenchymal transition (EMT), migration and invasion of thyroid tumor cells [27]. In PC3 prostate cancer cells autocrine TGFβ1 signalling appears to be involved in hypoxia-mediated secretion of the vascular endothelial growth factor A (VEGFA). Darrington et al. suggested a working model to explain the connections between hypoxia, TGFβ1 and VEGFA signalling: PC3 prostate cancer cells express VEGFR-2 protein that enables an autocrine VEGFA signalling. The VEGFA secretion is induced by both hypoxia and TGFβ1 and results in increased migration of PC3 cells. Furthermore, hypoxia directly stimulates TGFβ1 expression, which constitutes an autocrine mechanism to enhance the effects of hypoxia on VEGFA expression [28]. The influence of VEGFA on tumor cell migration has also been investigated in melanoma cells. In 2005, Lacal et al. analyzed the migratory behavior of human melanoma cells that simultaneously
] (]]]]) ]]]–]]]
5
produce VEGFA and express VEGFRs. In these cells a VEGFAdriven autocrine loop is responsible for their increased invasiveness to the extracellular matrix (ECM) compared to melanoma cells not expressing either VEGFA or VEGFRs [29]. In addition to the VEGFR/VEGFA system, the EGFR signalling may contribute to the tumorigenic potential of human melanoma cells. EGFR expression or overexpression has been proven in many melanoma cells as well as the secretion of different growth factors that activate EGFR, such as TGFα, EGF, amphiregulin, betacellulin and HB-EGF [30–36]. Meierjohann et al. could show that the mutationally activated EGFR variant Xmrk, which is responsible for the development of highly malignant tumors in Xiphophorus fish, leads to a clear increase of pigment cell motility. EGF stimulation of Xmrk induces the formation of a receptor/Fyn/ focal adhesion kinase (FAK) complex, activation of FAK, and increased focal contact and actin cytoskeleton turnover [37]. Advanced studies indicated that Xmrk mediates the production of biologically active secreted EGFR ligands. These ligands are able to enhance the activation of already pre-dimerized receptors in an autocrine manner [38]. In the present study we supposed an autocrine activation of EGFR to be involved in the regulation of pancreatic cancer cell migration. We demonstrated that CFPAC1 and IMIM-PC2 human pancreatic cancer cells express high levels of both EGFR and ErbB2 and only low levels of ErbB3 (Fig. 1). Similar to our results Ioannou et al. reported in the past that seven pancreatic cancer cell lines are positive for EGFR and ErB2, negative for ErbB4 and express extremely low or undetectable levels of ErbB3 [39]. Besides, several studies revealed an expression or even an overexpression of EGF, EGFR or the combination of EGF and EGFR in pancreatic carcinomas [40–42]. Furthermore, Murphy et al. identified an EGFR dependent autocrine growth pathway in pancreatic cancer cells [21]: when pancreatic cancer cells were grown without change of medium, proliferation was greater than when either medium was replaced frequently or cells were grown in the presence of the EGF receptor tyrosine kinase inhibitor AG1478. As a consequence of this, the authors suggest that soluble endogenous EGF-like growth factors mediate an autocrine EGFR activation and thus stimulate the proliferation of pancreatic cancer cells [21]. In light of these findings we hypothesized that autocrine EGFR and/or ErbB2 activation might be the reason for the constitutive PLCγ activation in pancreatic cancer cells. According to this model, EGFR and/or ErbB2 should be already activated in the cells without any exogenous growth factor treatment. Therefore, we performed migratory experiments with AG1478, the ErbB2 inhibitor 4-(3-Phenoxyphenyl)-5-cyano-2H-1,2,3-triazole and a neutralizing anti-EGFR antibody. All three substances were able to significantly inhibit the spontaneous migratory activity of CFPAC1 and IMIM-PC2 cells (Figs. 2 and 3), indicating a constitutive autocrine activation of EGFR and ErbB2. As no direct ligand for ErbB2 has yet been discovered and ErbB2 plays a role in the potentiation of ErbB receptor signalling as the preferred heterodimerization partner of all other ErbB family members [43–46], we focused the following experiments on EGFR signalling. Treatment with exogenous EGF increased the migratory activity of both pancreatic cancer cell lines (Fig. 3), suggesting that autocrine activation via the endogenously produced EGF was under our cell culture condition not maximally stimulating cells. Because of this induction of the migratory activity by exogenous EGF and the constitutive activation of EGFR potentially caused by endogenous
Please cite this article as: A.-M. Stock, et al., Induction of pancreatic cancer cell migration by an autocrine epidermal growth factor receptor activation, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.04.022
6
324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383
E XP E RI ME N TAL CE L L R ES E ARC H
EGF, we used an EGF immunoassay to quantify the EGF levels in the cell culture supernatants. Over a period of four days we could measure a continuous increase of the EGF concentration in the cell culture supernatants of both cell lines (Fig. 4), providing the direct evidence that CFPAC1 and IMIM-PC2 cells release EGF. This EGF secretion is a likely explanation for the constitutive EGFR activation, which leads to the previously described constitutive PLCγ phosphorylation [10]. As a consequence, the pancreatic cancer cells exhibit a very high spontaneous migratory activity that enables a high frequency of early invasion and metastasis development. Additional EGFR ligands different from EGF might contribute to this autocrine effect, too. However, these ligands have not been studied herein. To date, several antibodies and tyrosine kinase inhibitors targeting EGFR or ErbB2 have been approved for treatment of different human cancers: the anti-EGFR monoclonal antibody cetuximab for metastatic colorectal cancer, head and neck cancer; the anti-EGFR monoclonal antibody panitumumab for metastatic colorectal cancer; the EGFR tyrosine kinase inhibitors gefitinib and erlotinib for non-small cell lung cancer; the anti-ErbB2 monoclonal antibody trastuzumab for metastatic stomach cancers and breast cancer and the anti-ErbB2 monoclonal antibody pertuzumab as well as the dual EGFR/ErbB2 tyrosine kinase inhibitor lapatinib for breast cancer [47–50]. For the treatment of patients with pancreatic cancer only the EGFR tyrosine kinase inhibitor erlotinib has gained the FDA approval in combination with the chemotherapeutic drug gemcitabine [51]. EGFR and ErbB2 are not the only receptor tyrosine kinases, which might be responsible for the high spontaneous migration of pancreatic cancer cells via induction of PLCγ phosphorylation. As introduced before, the PLCγ is also a substrate for PDGF receptor tyrosine kinases [22,23]. Overexpression of PDGF ligands and receptors has been reported for many solid tumors including glioblastoma, meningioma, melanoma, ovarian cancer, prostate cancer, lung cancer, gastric carcinoma and pancreatic cancer [52]. Kawaguchi et al. studied the involvement of PDGF signalling in AsPC1 and BxPC3 pancreatic cancer cell migration and hypothesized that the homodimer PDGF-BB strongly induces cell migration via the Akt pathway [53]. In the present study flow cytometric analyses revealed a low expression of PDGFRα and no detectable amounts of PDGFRβ in IMIM-PC2 and CFPAC1 pancreatic cancer cells (Fig. 1). In subsequent migratory experiments a neutralizing anti-PDGFRα antibody significantly reduced the migratory activity of IMIM-PC2 cells. In CFPAC1 cells the antibody did not cause any significant effects (Fig. 5). Therefore, we assume that a PDGFRα activation contributes to the autocrine stimulation in IMIM-PC2 cells, but not in CFPAC1 cells. Further growth factor receptors or cytokine receptors might be involved, which were not investigated in this study.
Conclusion We suggest that autocrine EGFR activation by secreted EGF induces constitutive PLCγ phosphorylation and finally results in a high spontaneous migratory activity of pancreatic cancer cells. Besides this, the migratory experiments with the neutralizing PDGFRα antibody show the individuality of each cell line: In IMIM-PC2 cells PDGFRα seems to be involved, too, in CFPAC1 cells
] (]]]]) ]]]–]]]
not. Thus, our results and the literature discussed herein provide the evidence for the importance of growth factors and their receptors as targets for the development of anti-metastatic drugs, which have to be individually adjusted to the patients.
Acknowledgments This work was supported by the Fritz Bender Foundation (Munich, Q2 Germany).
references [1] T. Schnelldorfer, A.L. Ware, M.G. Sarr, T.C. Smyrk, L. Zhang, R. Qin, R.E. Gullerud, J.H. Donohue, D.M. Nagorney, M.B. Farnell, Longterm survival after pancreatoduodenectomy for pancreatic adenocarcinoma: is cure possible?, Ann. Surg. 247 (2008) 456–462. [2] J. Guo, W. Lou, Y. Ji, S. Zhang, Effect of CCR7, CXCR4 and VEGF-C on the lymph node metastasis of human pancreatic ductal adenocarcinoma, Oncol. Lett. 5 (2013) 1572–1578. [3] F. Entschladen, T.L.t. Drell, K. Lang, J. Joseph, K.S. Zänker, Tumourcell migration, invasion, and metastasis: navigation by neurotransmitters, Lancet Oncol. 5 (2004) 254–258. [4] D. Kedrin, J. van Rheenen, L. Hernandez, J. Condeelis, J.E. Segall, Cell motility and cytoskeletal regulation in invasion and metastasis, J. Mammary Gland Biol. Neoplasia 12 (2007) 143–152. [5] F. Balkwill, A. Mantovani, Inflammation and cancer: back to Virchow?, Lancet 357 (2001) 539–545. [6] F. Entschladen, K.S. Zänker, D.G. Powe, Heterotrimeric G protein signaling in cancer cells with regard to metastasis formation, Cell Cycle 10 (2011) 1086–1091. [7] F. Entschladen, T.L.t. Drell, K. Lang, J. Joseph, K.S. Zänker, Neurotransmitters and chemokines regulate tumor cell migration: potential for a new pharmacological approach to inhibit invasion and metastasis development, Curr. Pharm. Des. 11 (2005) 403–411. [8] P. Bastian, A. Balcarek, C. Altanis, C. Strell, B. Niggemann, K.S. Zaenker, F. Entschladen, The inhibitory effect of norepinephrine on the migration of ES-2 ovarian carcinoma cells involves a Rap1-dependent pathway, Cancer Lett. 274 (2009) 218–224. [9] A.M. Stock, G. Troost, B. Niggemann, K.S. Zanker, F. Entschladen, Targets for anti-metastatic drug development, Curr. Pharm. Des. (2013). [10] A.M. Stock, D.G. Powe, S.A. Hahn, G. Troost, B. Niggemann, K.S. Zanker, F. Entschladen, Norepinephrine inhibits the migratory activity of pancreatic cancer cells, Exp. Cell Res. 319 (2013) 1744–1758. [11] B. Niggemann, T.L.t. Drell, J. Joseph, C. Weidt, K. Lang, K.S. Zaenker, F. Entschladen, Tumor cell locomotion: differential dynamics of spontaneous and induced migration in a 3D collagen matrix, Exp. Cell Res. 298 (2004) 178–187. [12] C. Strell, B. Niggemann, M.J. Voss, D.G. Powe, K.S. Zanker, F. Entschladen, Norepinephrine promotes the beta1-integrinmediated adhesion of MDA-MB-231 cells to vascular endothelium by the induction of a GROalpha release, Mol. Cancer Res. 10 (2012) 197–207. [13] D.L. Cadena, G.N. Gill, Receptor tyrosine kinases, FASEB J. 6 (1992) 2332–2337. [14] G. Alonso, M. Koegl, N. Mazurenko, S.A. Courtneidge, Sequence requirements for binding of Src family tyrosine kinases to activated growth factor receptors, J. Biol. Chem. 270 (1995) 9840–9848. [15] N.R. Lemoine, C.M. Hughes, C.M. Barton, R. Poulsom, R.E. Jeffery, G. Kloppel, P.A. Hall, W.J. Gullick, The epidermal growth factor receptor in human pancreatic cancer, J. Pathol. 166 (1992) 7–12.
Please cite this article as: A.-M. Stock, et al., Induction of pancreatic cancer cell migration by an autocrine epidermal growth factor receptor activation, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.04.022
E X PE R IM EN TA L C ELL R E S EA RC H
384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443
[16] K. Kimura, T. Sawada, M. Komatsu, M. Inoue, K. Muguruma, T. Nishihara, Y. Yamashita, N. Yamada, M. Ohira, K. Hirakawa, Antitumor effect of trastuzumab for pancreatic cancer with high HER-2 expression and enhancement of effect by combined therapy with gemcitabine, Clin. Cancer Res. 12 (2006) 4925–4932. [17] M. Ebert, M. Yokoyama, H. Friess, M.S. Kobrin, M.W. Buchler, M. Korc, Induction of platelet-derived growth factor A and B chains and over-expression of their receptors in human pancreatic cancer, Int. J. Cancer 62 (1995) 529–535. [18] Y. Yarden, M.X. Sliwkowski, Untangling the ErbB signalling network, Nat. Rev. Mol. Cell Biol. 2 (2001) 127–137. [19] A. Citri, Y. Yarden, EGF-ERBB signalling: towards the systems level, Nat. Rev. Mol. Cell Biol. 7 (2006) 505–516. [20] P. Seshacharyulu, M.P. Ponnusamy, D. Haridas, M. Jain, A.K. Ganti, S.K. Batra, Targeting the EGFR signaling pathway in cancer therapy, Expert Opin. Ther. Targets 16 (2012) 15–31. [21] L.O. Murphy, M.W. Cluck, S. Lovas, F. Otvos, R.F. Murphy, A.V. Schally, J. Permert, J. Larsson, J.A. Knezetic, T.E. Adrian, Pancreatic cancer cells require an EGF receptor-mediated autocrine pathway for proliferation in serum-free conditions, Br. J. Cancer 84 (2001) 926–935. [22] H.K. Kim, J.W. Kim, A. Zilberstein, B. Margolis, J.G. Kim, J. Schlessinger, S.G. Rhee, PDGF stimulation of inositol phospholipid hydrolysis requires PLC-gamma 1 phosphorylation on tyrosine residues 783 and 1254, Cell 65 (1991) 435–441. [23] J. Meisenhelder, P.G. Suh, S.G. Rhee, T. Hunter, Phospholipase C-gamma is a substrate for the PDGF and EGF receptor proteintyrosine kinases in vivo and in vitro, Cell 57 (1989) 1109–1122. [24] K. Masur, K. Lang, B. Niggemann, K.S. Zänker, F. Entschladen, PKC High, alpha and low E-cadherin expression contribute to high migratory activity of colon carcinoma cells, Mol. Biol. Cell 12 (2001) 1973–1982. [25] D. Hanahan, R.A. Weinberg, Hallmarks of cancer: the next generation, Cell 144 (2011) 646–674. [26] R. Hasina, K. Matsumoto, N. Matsumoto-Taniura, I. Kato, M. Sakuda, T. Nakamura, Autocrine and paracrine motility factors and their involvement in invasiveness in a human oral carcinoma cell line, Br. J. Cancer 80 (1999) 1708–1717. [27] G. Riesco-Eizaguirre, I. Rodriguez, A. De la Vieja, E. Costamagna, N. Carrasco, M. Nistal, P. Santisteban, The BRAFV600E oncogene induces transforming growth factor beta secretion leading to sodium iodide symporter repression and increased malignancy in thyroid cancer, Cancer Res. 69 (2009) 8317–8325. [28] E. Darrington, M. Zhong, B.H. Vo, S.A. Khan, Vascular endothelial growth factor A, secreted in response to transforming growth factor-beta1 under hypoxic conditions, induces autocrine effects on migration of prostate cancer cells, Asian J. Androl. 14 (2012) 745–751. [29] P.M. Lacal, F. Ruffini, E. Pagani, S. D’Atri, An autocrine loop directed by the vascular endothelial growth factor promotes invasiveness of human melanoma cells, Int. J. Oncol. 27 (2005) 1625–1632. [30] K. Krasagakis, C. Garbe, C.C. Zouboulis, C.E. Orfanos, Growth control of melanoma cells and melanocytes by cytokines, Recent Results Cancer Res. 139 (1995) 169–182. [31] B. Singh, M. Schneider, P. Knyazev, A. Ullrich, UV-induced EGFR signal transactivation is dependent on proligand shedding by activated metalloproteases in skin cancer cell lines, Int. J. Cancer 124 (2009) 531–539. [32] N. Bardeesy, M. Kim, J. Xu, R.S. Kim, Q. Shen, M.W. Bosenberg, W.H. Wong, L. Chin, Role of epidermal growth factor receptor signaling in RAS-driven melanoma, Mol. Cell Biol. 25 (2005) 4176–4188. [33] L.A. Akslen, H. Puntervoll, I.M. Bachmann, O. Straume, E. Vuhahula, R. Kumar, A. Molven, Mutation analysis of the EGFR-NRAS-BRAF pathway in melanomas from black Africans
[34]
[35]
[36]
[37]
[38]
[39]
[40]
[41]
[42]
[43]
[44]
[45]
[46]
[47]
[48]
[49]
[50]
] (]]]]) ]]]–]]]
7
and other subgroups of cutaneous melanoma, Melanoma Res. 18 (2008) 29–35. P.E. de Wit, S. Moretti, P.G. Koenders, M.A. Weterman, G.N. van Muijen, B. Gianotti, D.J. Ruiter, Increasing epidermal growth factor receptor expression in human melanocytic tumor progression, J. Invest. Dermatol. 99 (1992) 168–173. S. Mattei, M.P. Colombo, C. Melani, A. Silvani, G. Parmiani, M. Herlyn, Expression of cytokine/growth factors and their receptors in human melanoma and melanocytes, Int. J. Cancer 56 (1994) 853–857. L.E. Sparrow, P.J. Heenan, Differential expression of epidermal growth factor receptor in melanocytic tumours demonstrated by immunohistochemistry and mRNA in situ hybridization, Australas. J. Dermatol. 40 (1999) 19–24. S. Meierjohann, E. Wende, A. Kraiss, C. Wellbrock, M. Schartl, The oncogenic epidermal growth factor receptor variant Xiphophorus melanoma receptor kinase induces motility in melanocytes by modulation of focal adhesions, Cancer Res. 66 (2006) 3145–3152. J.A. Laisney, T.D. Mueller, M. Schartl, S. Meierjohann, Hyperactivation of constitutively dimerized oncogenic EGF receptors by autocrine loops, Oncogene 32 (2013) 2403–2411. N. Ioannou, A.G. Dalgleish, A.M. Seddon, D. Mackintosh, U. Guertler, F. Solca, H. Modjtahedi, Anti-tumour activity of afatinib, an irreversible ErbB family blocker, in human pancreatic tumour cells, Br. J. Cancer 105 (2011) 1554–1562. K. Uegaki, Y. Nio, Y. Inoue, Y. Minari, Y. Sato, M.M. Song, M. Dong, K. Tamura, Clinicopathological significance of epidermal growth factor and its receptor in human pancreatic cancer, Anticancer Res. 17 (1997) 3841–3847. M. Bloomston, A. Bhardwaj, E.C. Ellison, W.L. Frankel, Epidermal growth factor receptor expression in pancreatic carcinoma using tissue microarray technique, Dig. Surg. 23 (2006) 74–79. K. Tobita, H. Kijima, S. Dowaki, H. Kashiwagi, Y. Ohtani, Y. Oida, H. Yamazaki, M. Nakamura, Y. Ueyama, M. Tanaka, S. Inokuchi, H. Makuuchi, Epidermal growth factor receptor expression in human pancreatic cancer: Significance for liver metastasis, Int. J. Mol. Med. 11 (2003) 305–309. E. Tzahar, H. Waterman, X. Chen, G. Levkowitz, D. Karunagaran, S. Lavi, B.J. Ratzkin, Y. Yarden, A hierarchical network of interreceptor interactions determines signal transduction by Neu differentiation factor/neuregulin and epidermal growth factor, Mol. Cell. Biol. 16 (1996) 5276–5287. D. Graus-Porta, R.R. Beerli, J.M. Daly, N.E. Hynes, ErbB-2, the preferred heterodimerization partner of all ErbB receptors, is a mediator of lateral signaling, EMBO J. 16 (1997) 1647–1655. R.R. Beerli, D. Graus-Porta, K. Woods-Cook, X. Chen, Y. Yarden, N.E. Hynes, Neu differentiation factor activation of ErbB-3 and ErbB-4 is cell specific and displays a differential requirement for ErbB-2, Mol. Cell. Biol. 15 (1995) 6496–6505. D. Graus-Porta, R.R. Beerli, N.E. Hynes, Single-chain antibodymediated intracellular retention of ErbB-2 impairs Neu differentiation factor and epidermal growth factor signaling, Mol. Cell. Biol. 15 (1995) 1182–1191. N. Ioannou, A.M. Seddon, A. Dalgleish, D. Mackintosh, H. Modjtahedi, Expression pattern and targeting of HER family members and IGF-IR in pancreatic cancer, Front. Biosci. 17 (2012) 2698– 2724. C.M. Rocha-Lima, L.E. Raez, Erlotinib (tarceva) for the Treatment of Non-small-cell Lung Cancer and Pancreatic Cancer, P T 34 (2009) 554–564. K.S. Zaenker, F. Entschladen, Paving roads for new drugs in oncology, Recent Patents Anticancer Drug Discovery 4 (2009) 137–145. N. Ioannou, A.M. Seddon, A. Dalgleish, D. Mackintosh, H. Modjtahedi, Treatment with a combination of the ErbB (HER) family blocker afatinib and the IGF-IR inhibitor, NVP-AEW541 induces
Please cite this article as: A.-M. Stock, et al., Induction of pancreatic cancer cell migration by an autocrine epidermal growth factor receptor activation, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.04.022
8
444 445 446 447 448 449 450 451
E XP E RI ME N TAL CE L L R ES E ARC H
synergistic growth inhibition of human pancreatic cancer cells, BMC Cancer 13 (2013) 41. [51] R.K. Kelley, A.H. Ko, Erlotinib in the treatment of advanced pancreatic cancer, Biologics 2 (2008) 83–95. [52] A. Ostman, C.H. Heldin, Involvement of platelet-derived growth factor in disease: development of specific antagonists, Adv. Cancer Res. 80 (2001) 1–38.
] (]]]]) ]]]–]]]
[53] J. Kawaguchi, S. Adachi, I. Yasuda, T. Yamauchi, T. Yoshioka, M. Itani, O. Kozawa, H. Moriwaki, UVC irradiation suppresses platelet-derived growth factor-BB-induced migration in human pancreatic cancer cells, Oncol. Rep. 27 (2012) 935–939.
Please cite this article as: A.-M. Stock, et al., Induction of pancreatic cancer cell migration by an autocrine epidermal growth factor receptor activation, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.04.022
