Activation of the EGFR Gene Target Epha2 Inhibits Epidermal Growth Factor–Induced Cancer Cell Motility

Activation of the EGFR Gene Target EphA2 Inhibits Epidermal Growth Factor–Induced Cancer Cell Motility

Alice Bjerregaard Larsen,1 Mikkel Wandahl Pedersen,1 Marie-The´re´se Stockhausen,1 Michael Vibo Grandal,2 Bo van Deurs,2 and Hans Skovgaard Poulsen1

1Department of Radiation Biology, Section 6321, Copenhagen University Hospital and 2Department of Cellular and Molecular Medicine, The Panum Building, The Panum Institute, University of Copenhagen, Copenhagen, Denmark

Abstract The cDNA sequence of EphA2 (previously known as EphA2 overexpression has been reported in many epithelial cell kinase) was first described by Lindberg and cancers and is believed to playan important role in tumor Hunter in 1990 (10). EphA2 belongs to the Eph receptor family, metastasis and angiogenesis. We show that the activated which is the largest subfamily of receptor tyrosine kinases (11). epidermal growth factor receptor (EGFR) and the Based on similarities in the extracellular domain sequences, the cancer-specific constitutivelyactive EGFR type III deletion Eph receptors are divided into two classes, EphA and EphB. mutant (EGFRvIII) induce the expression of EphA2 in The Eph receptors bind cell membrane–anchored ligands, mammalian cell lines, including the human cancer cell known as ephrins, which are divided into two classes, ephrin-A lines A431 and HN5. The regulation is partiallydependent and ephrin-B. The ephrin-A class are associated to the on downstream activation of mitogen-activated protein membrane by a glycosylphosphatidylinositol anchor whereas kinase/extracellular signal–regulated kinase kinase and the ephrin-B class are transmembrane proteins (12). Generally, is a direct effect on the EphA2 promoter. Furthermore, EphA receptors bind to ephrin-A ligands and EphB receptors to EGFR and EphA2 both localize to the plasma membrane ephrin-B ligands, although a few exceptions exist (13). and EphA2 coimmunoprecipitates with activated EGFR Interaction between an Eph receptor and its ligand results in and EGFRvIII. Ligand activation of EphA2 and EphA2 heterodimers that, by clustering, form heterotetramers (12), knockdown bysmall interfering RNA inhibit EGF-induced leading to transphosphorylation of tyrosine residues of the cell motilityof EGFR-overexpressing human cancer cells, juxtamembrane domain and to activation of the tyrosine kinase indicating a functional role of EphA2 in EGFR-expressing (14, 15). cancer cells. (Mol Cancer Res 2007;5(3):283–93) The Eph family of receptors and their ligands are expressed in adult human tissues and in the developing nervous system Introduction (16). These receptor-ligand systems are involved in cell-cell repulsion and attraction and play a role in tissue patterning, The epidermal growth factor (EGF) and its receptor (EGFR) neuronal targeting, and angiogenesis during embryonic devel- constitute a well-characterized receptor-ligand system that plays opment (17-20). In addition, EphA2 overexpression has been an important role in the regulation of cell growth, proliferation, survival, and motility (1-4). EGFR is involved in oncogenic reported in many cancers including those of the breast, lung, transformation, which can be caused by receptor overexpres- esophagus, stomach, cervix, ovary, colon, prostate, kidney, and sion, autocrine ligand loops, gene amplification, or activating skin (21-29). Overexpression of EphA2 has been correlated mutations such as the EGFR type III deletion mutant with increased metastatic potential and decreased survival (EGFRvIII; refs. 5-8). (22-24, 27, 29). Furthermore, increasing evidence suggests that In a global search for EGFR and EGFRvIII regulated genes, EphA2 is involved in tumor angiogenesis (26, 30, 31). we recently found the mRNA level of the receptor tyrosine Little is known about the regulation of EphA2 expression. kinase EphA2 to be up-regulated by ligand-activated EGFR and However, members of the p53 family of transcription factors the constitutively active variant EGFRvIII (9). have been shown to increase EphA2 transcription (32). High levels of EphA2 have also been observed in cells with overactive RAS signaling (33-35). Received 9/27/06; revised 12/18/06; accepted 1/9/07. Understanding the regulation and function of EphA2 in Grant support: Danish Cancer Society, the Danish Medical Research Council, cancer cells may lead to therapeutics for several types of the University of Copenhagen, the Novo Nordic Foundation, the John and Birthe aggressive cancers. Therefore, we are interested in the Meyer Foundation, and the Danish Cancer Research Foundation. The costs of publication of this article were defrayed in part by the payment of mechanisms that control the regulation and the function of page charges. This article must therefore be hereby marked advertisement in EphA2 in cancer cells. The aim of this study was to investigate accordance with 18 U.S.C. Section 1734solely to indicate this fact. the regulation of EphA2 by ligand-activated EGFR and by the Requests for reprints: Hans Skovgaard Poulsen, Department of Radiation Biology, The Finsen Center, Section 6321, Copenhagen University Hospital, constitutively active EGFRvIII. Furthermore, we wanted to Blegdamsvej 9, DK-2100 Copenhagen, Denmark. Phone: 45-35-456303; Fax: 45- investigate the significance of EphA2 up-regulation in EGFR- 35-456301. E-mail: [email protected] Copyright D 2007 American Association for Cancer Research. expressing cells by analyzing the effect of ligand stimulation doi:10.1158/1541-7786.MCR-06-0321 on EphA2 localization and EGF-induced cancer cell migration.

We show that activated EGFR and EGFRvIII rapidly stimulate EphA2 expression in several mammalian cell lines including the human cancer cell lines A431 and HN5, which both naturally overexpress EGFR. The stimulation of EphA2 expression is dependent on the tyrosine kinase activity of EGFR/EGFRvIII and partially on the activation of mitogen- activated protein kinase/extracellular signal–regulated kinase kinase (MEK). Our results further show that EphA2 and EGFR localize to the plasma membrane and that ligand stimulation of EphA2 or EphA2 knockdown by small interfering RNA (siRNA) affects the EGF-induced cell motility by inhibiting cell migration and modulating the migration pattern to a less uniform and more scattered type. These results indicate that EphA2 could play a significant role in regulating EGF-induced migration of human cancer cells.

Results Activation of EGFR Increases EphA2 mRNA and Protein Levels Recently, we have investigated changes in the transcriptome induced by ligand activation of EGFR or the constitutively active mutant EGFRvIII in mouse fibroblast cell lines expressing either wild-type EGFR (NR6wtEGFR) or EGFRvIII (NR6M) using Affymetrix oligonucleotide arrays (9). One of the genes, whose expression was found to be induced by ligand- activated EGFR and by EGFRvIII, encodes the tyrosine kinase receptor EphA2. To confirm and extend these results in human cancer cells, time profiles of the EGFR-induced expression of EphA2 were investigated in the human head and neck carcinoma cell line HN5 and in the human epidermoid carcinoma cell line A431, both overexpressing EGFR. Serum-starved cells were stimulated with EGF for up to 24h and processed for quantitative real-time reverse transcription-PCR (Fig. 1A). In both cell lines, a maximum increase in EphA2 mRNA levels was observed after 2 h of EGF stimulation. A 5-fold increase in EphA2 mRNA level was detected in the A431 cell line whereas a 9-fold increase was seen in HN5 cells (Fig. 1A). To verify that EphA2 protein levels are induced by EGFR activation, serum-starved A431, HN5, NR6, and NR6wtEGFR cells were stimulated with EGF for up to 48 h and EphA2 protein levels were analyzed (Fig. 1B). An increase in EphA2 protein levels was detected in the EGFR-expressing cell lines after 6 h of EGF stimulation and the level remained high during the 48-h period. A similar increase in EphA2 protein levels was seen when releasing the cell line expressing constitutive activated EGFRvIII (NR6M) from AG1478 inhibition, thus allowing receptor autophosphorylation (Fig. 1B). Quantification of the induced EphA2 protein levels in the FIGURE 1. A. Serum-starved A431 and HN5 cells were stimulated EGFR-expressing cell lines showed that EGF stimulation with 10 nmol/L EGF for the indicated periods. EphA2 mRNA levels were increased the protein levels of EphA2 three to five times with detected in Trizol-extracted RNA samples by quantitative real-time reverse a doubling time ranging from 3.0 to 4.3 h (Fig. 1C). Similarly, transcription-PCR analyses. Points, average of three to four independent experiments; bars, SE. B. Western blot analysis showing levels of EphA2 releasing the NR6M cell line from AG1478 inhibition in serum-starved NR6, NR6wtEGFR, A431, and HN5 cells stimulated with increased the protein levels of EphA2 with a doubling time 10 nmol/L EGF for indicated periods and in NR6M cells inhibited with 5 of 3.9 h (Fig. 1C). EGF stimulation of the parental NR6 cell Amol/L AG1478 for 48 h and then released from inhibition at indicated periods. Tubulin levels confirm equal protein loading. Representative of line, which lacks endogenous EGFR expression, had no effect four independent experiments. C. Relative increases in EphA2 protein on the level of EphA2 (Fig. 1B and C). These results show that levels quantified by measuring band intensities detected by Western blot analysis, in which a representative experiment of four is shown in B. EGF stimulation is an important inducer of EphA2 mRNA and Bars, SE. protein levels in mammalian cell lines expressing EGFR

including two human cancer cell lines. Likewise, activation of investigate the effects of ligand stimulation of EGFR or EphA2 EGFRvIII similarly induces EphA2 expression. on the localization of both receptors. Cells were serum starved and treated with either EGF or soluble EphA2-ligand (ephrin- The EGFR-Stimulated Increase in EphA2 Protein Levels A1/Fc) for 48 h followed by fixation and indirect staining of Is Dependent on MEK Activity EphA2 and EGFR. The cells were investigated by confocal A panel of small molecular weight inhibitors were used to microscopy and images of A431 and HN5 cells revealed that identify which signaling pathways downstream of ligand- EGFR and EphA2 primarily localized to the cell membrane in activated EGFR might contribute to the EGFR-induced expres- untreated cells (Fig. 3A). EGF stimulation increased EphA2 sion of EphA2. The EGFR-specific inhibitors AG1478 and levels in both cell lines, and the receptor seemed to be gefitinib clearly decreased the EGFR- and EGFRvIII-induced distributed at the plasma membrane or intracellularly. EGF expression of EphA2 in the NR6wtEGFR, NR6M, and HN5 cell stimulation induced an almost complete loss of EGFR at the cell lines (Fig. 2A). However, AG1478 had no effect on the EphA2 membrane of A431 cells, and residual EGFR was found protein level in A431 cells. Detection of EGFR tyrosine intracellularly (Fig. 3A). A less pronounced response to EGF phosphorylation (Y1173) showed that AG1478 treatment had stimulation on EGFR localization was observed in the HN5 cell no effect on EGFR phosphorylation in A431 cells (not shown). line. Ephrin-A1 stimulation resulted in removal of EphA2 from The MEK-specific inhibitors U0126 and PD98059 had partial the plasma membrane without affecting EGFR localization. inhibitory effects on the EGF-induced increase in EphA2 level in the A431, NR6wtEGFR, and NR6M cell lines and a strong inhibitory effect in the HN5 cell line. Inhibitors of phosphati- dylinositol 3-kinase (wortmannin and LY294002), phospholi- pase Cg (U73122), p38 (SB203580), Janus-activated kinase-2 (AG490), and Src (PP2 and SU6656) had no effect on the EGFR/ EGFRvIII–induced expression of EphA2 (not shown). These results indicate that the EGFR- and EGFRvIII-induced expression of EphA2 is dependent on MEK activity; however, the degree of dependence seems to be cell type specific.

EGFR Activation Stimulates EphA2 Promoter Activity To determine if the EGFR-induced expression of EphA2 was through activation of the EphA2 promoter, a luciferase reporter assay was done. A human EphA2 promoter fragment spanning nucleotides À4030 to +21 relative to the putative EphA2 transcriptional start site, previously cloned into a luciferase reported vector (À4030-EphA2-Luc), was used (36). Initially, HN5 cells were transiently transfected with À4030-EphA2-Luc and stimulated with various concentrations of EGF and in different stimulation periods to determine the optimal conditions for stimulation of EphA2 promoter activity. A concentration of 5 nmol/L EGF and a stimulation period of 6 h were found to give the highest increase in EphA2 promoter activity (not shown) and were used in subsequent experiments. To test if the increase in EphA2 promoter activity was dependent on EGFR kinase activity, HN5 and A431 cells transiently transfected with 4030-EphA2-Luc were stimulated with EGF alone or in combination with AG1478 or gefitinib. EGF stimulation significantly increased the EphA2 promoter activity in both cell lines. However, the response was more pronounced in HN5 cells as compared with A431 cells, most likely due to the high basal activity level in A431 cells as shown by Western blotting (Fig. 2A). The EphA2 promoter activity significantly decreased in response to AG1478 and FIGURE 2. A. Western blot analysis showing EphA2 protein levels in gefitinib treatment (Fig. 2B). Thus, these results show that serum-starved NR6wtEGFR, NR6M, A431, and HN5 cells treated with 5 Amol/L AG1478, 2 Amol/L gefitinib, 10 Amol/L U0126, or 50 Amol/L EGFR activation induces expression of EphA2 in human cancer PD98059 and/or with 10 nmol/L EGF for 48 h. Tubulin levels confirm equal cell lines by a stimulatory effect on the EphA2 promoter. protein loading. B. Percent increase in luciferase activity of HN5 cells and A431 cells transiently transfected with À4030-EphA2-Luc and stimulated with 5 nmol/L EGF for 6 h in the presence or absence of EphA2 Localizes with EGFR at the Cell Membrane 5 Amol/L AG1478 or 1 Amol/L gefitinib. Representative data of two independent experiments done in duplicates and normalized to SV40 Overexpression of EphA2 has been shown to affect the promoter activity (internal control) for each condition. Bars, SE. *, P < 0.05, subcellular localization of EphA2 (37). Therefore, we wished to compared with unstimulated cells.

FIGURE 3. A. Representative confocal images of A431 and HN5 cells stained for EphA2 (green), EGFR (red), and nuclei (blue) incubated for 48 h with 10 nmol/L EGF or 1 Ag/mL ephrin-A1/Fc or left unstimulated. Bars, 20 Am. B. Scatter diagrams of pixel intensities of EphA2 and EGFR staining from the images of A431 cells shown in A. Red lines on axis, threshold for background pixels; blue lines, mean intensity of EphA2 or EGFR staining above the background threshold. C and D. Columns, mean staining intensities of EphA2 and EGFR in A431 cells (C) and HN5 cells (D) incubated for 48 h with 10 nmol/L EGF or 1 Ag/mL ephrin-A1/Fc or left unstimulated. More than 80 cells in 10 randomly selected areas of each sample were used for measuring the staining intensities in each setting. Bars, 95% confidence interval. *, P < 0.05; **, P < 0.001; ***, P < 0.00001, compared with unstimulated cells.

FIGURE 4. A. Immunoprecipitation with monoclonal anti-EGFR antibody and cell lysates from serum-starved cells (NR6, NR6wtEGFR, NR6M, A431, and HN5) left untreated or stimulated with 10 nmol/L EGF for 24 h. The immunoprecipitates were subjected to Western blot analysis with antibodies directed against EphA2, EGFR, or EGFRvIII. Results from one of two similar experiments. B. Western blot analysis showing levels of EphA2 and EGFR/EGFRvIII in immunoprecipitated samples using antiphosphotyrosine antibody (PY-20) of serum-starved NR6, NR6wtEGFR, NR6M, A431, and HN5 cells stimulated with 1 Ag/mL ephrin-A1/Fc or 10 nmol/L EGF for 15 min.

Occasionally, some EphA2 staining was seen in the nucleus be detected in the EGFR-negative cell line NR6 after (Fig. 3A). immunoprecipitation with EGFR antibody. Stimulation of The pixel intensities of EphA2 and EGFR staining in images NR6wtEGFR and A431 cells with EGF resulted in a clear of unstimulated, EGF-stimulated, and ephrin-A1–stimulated increase in coprecipitated EphA2, whereas EphA2 coprecipi- cells were extracted and processed with confocal microscope tated with EGFRvIII irrespective of EGF stimulation in the associated software. Scatter diagrams with pixel intensities of NR6M cell line expressing constitutively active EGFRvIII (Fig. EGFR staining and EphA2 staining from the images of 4A). These results indicate that EGFR/EGFRvIII activation is unstimulated and EGF-stimulated A431 cells seen in Fig. 3A required for EphA2 coimmunoprecipitation with EGFR/EGFR- are illustrated in Fig. 3B. By examining the pixel distribution in vIII. The level of EGFR phosphorylation in the immunopreci- the scatter diagrams and the mean staining intensities, it is seen pitated samples was detected by Western blot analysis with that EGF stimulation resulted in increased EphA2 staining and antibody against phosphorylated tyrosine (PY-20; not shown). decreased EGFR staining (Fig. 3B). In agreement with this, by The results showed high phosphorylation level in HN5 cells calculating the mean staining intensities of EphA2 and EGFR independent of EGF stimulation, possibly explaining why staining from images with low magnification and settings EphA2 coimmunoprecipitated with EGFR independent of EGF optimal for quantification, the levels of EphA2 staining were stimulation in this cell line (Fig. 4A). found to increase 2.2- and 1.6-fold on EGF stimulation in the To investigate if the association between EGFR and EphA2 A431 and HN5 cell lines, respectively (Fig. 3C and D). leads to receptor transphosphorylation, cells were stimulated The levels of EGFR staining decreased 1.9- and 1.3-fold in the with either EGF or ephrin-A1/Fc. The effect of EGF and A431 and HN5 cell lines, respectively (Fig. 3C and D). ephrin-A1/Fc on either EphA2 or EGFR tyrosine phosphory- The changes in EGFR and EphA2 staining intensities were lation was analyzed by immunoprecipitation of tyrosine- statistically significant (P < 0.05-0.00001). Ephrin-A1 stimula- phosphorylated proteins followed by detection of EGFR and tion slightly decreased the levels of EphA2 and EGFR in both EphA2 by Western blot analysis (Fig. 4B). cell lines, but this was not statistically significant (Fig. 3C and The results showed that 15 min of EGF stimulation induced D). Quantification of staining intensities from images acquired phosphorylation of EGFR and that 15 min of ephrin-A1/Fc with high magnification and settings optimal for visual stimulation induced EphA2 phosphorylation, but either ligand presentation gave similar results (not shown). These results failed to induce tyrosine phosphorylation of the other receptor show that EphA2 and EGFR both localize to the plasma (Fig. 4B). Preliminary experiments showed that EphA2 and membrane of cancer cells and that EGF stimulation increases the EGFR did not coimmunoprecipitate after EGF stimulation for level of EphA2 staining and induces EGFR down-regulation. 15 min. Furthermore, EGF stimulation for 24h induced EphA2 phosphorylation whereas ephrin-A1/Fc had no effect on EGFR Activated EGFR and EGFRvIII Associate with EphA2 phosphorylation (not shown). Thus, these data suggest that Based on the observed EphA2 and EGFR colocalization at the EGFR and EphA2 associate after prolonged EGF stimulation cell membrane, we speculated whether an interaction between and that the outcome of this association could involve EGFR and EphA2 could be detected by coimmunoprecipitation transphosphorylation of EphA2. and whether the phosphorylation status of EGFR could influence this association. Coimmunoprecipitation experiments were EphA2 Activation Modulates EGF-Induced Motility carried out using NR6, NR6M, NR6wtEGFR, A431, and HN5 EGFR activation is known to stimulate cell proliferation and cells (Fig. 4A). Serum-starved cells were left untreated or motility (1, 2). Therefore, we inquired if activation of EphA2 stimulated with EGF for 24h and EGFR was immunoprecipi- could influence EGF-induced proliferation and motility of tated from the cellular lysates. Neither EGFR nor EphA2 could EGFR-expressing cells. Ligand activation of EphA2 did not

have an effect on EGF-induced proliferation (not shown) but sc-siRNA (P < 0.001). Importantly, these results show that inhibited EGF-induced motility (Fig. 5A-D). The motility of knockdown of EphA2 results in reduced EGF-induced migra- HN5 cells was investigated by a wound healing assay (Fig. 5A tion and indicate that EphA2 receptors, in the absence of ephrin- and B) and a three-dimensional spheroid motility assay (Fig. 5C A1/Fc stimulation, stimulate EGF-induced cell motility. and D). In both assays, ephrin-A1/Fc stimulation of HN5 cells inhibited EGF-induced motility. Quantification showed that Discussion ephrin-A1/Fc decreased EGF-induced wound closure by 41% In the present study, we report that activated EGFR and after 24h ( P < 0.0001) and 30% after 48 h (P < 0.001; Fig. 5B). EGFRvIII induce expression of EphA2 and that this regulation Intriguingly, we found that ephrin-A1/Fc stimulation of HN5 seems to involve downstream activation of the MEK signaling spheroids not just reduced the EGF-induced migration of HN5 pathway and stimulation of EphA2 promoter activity. Further- cells out from the spheroids but also altered the motility to a less more, we found that EphA2 localizes with EGFR at the plasma uniform and more scattered type (Fig. 5C). This effect was not membrane and that the two receptors can be coimmunopreci- seen in the wound healing assay and suggests that the effect of pitated. Our results further show that ligand stimulation of EphA2 ligand stimulation on cell motility depends on the three- EphA2 or EphA2 knockdown by siRNA inhibits EGF-induced dimensional organization of the cells. To further investigate the motility of human cancer cells overexpressing EGFR. These role of EphA2 in EGF-induced motility, the effect of EphA2 observations indicate that EphA2 plays a role in EGFR- knockdown by siRNA was investigated by a wound healing mediated cancer cell motility. assay (Fig. 5E and F). Quantification of the migration distance (wound closure) showed that the EGF-induced wound closure Activated EGFR Induces Expression of EphA2 was significantly decreased in EphA2 knockdown cells EGF stimulation was found to increase EphA2 mRNA and compared with the negative control cells transfected with protein levels in a number of EGFR-expressing cell lines

FIGURE 5. A. Wound healing assay using confluent serum-starved HN5 cells gently wounded through the central axis. Cells were stimulated with 2 Ag/mL ephrin-A1/Fc and/or 1 nmol/L EGF for 48 h. Images were taken at 0, 24, and 48 h. B. Quantification of cell motility by measuring the distance between the invading front of cells in six random selected microscopic fields for each condition and time point. The degree of motility is expressed as percent of wound closure as compared with the zero time point. C. Spheroid motility assay using spheroids of HN5 cells allowed to adhere and migrate in the presence of 2 Ag/mL ephrin-A1/Fc and/or 1 nmol/L EGF. Images were taken at 24 and 48 h with 4Â and 10Â objectives. D. Quantification of cell motility by measuring the distance between the edge of the spheroid and farthest migrated cell at three random selected areas for three spheroids for each condition and time point. The degree of motility is expressed as migrated distance compared with unstimulated cells. E. Wound healing assay using confluent HN5 cells transfected with 25 nmol/L siRNA targeting EphA2 (EphA2-siRNA) or 25 nmol/L negative control siRNA (sc-siRNA) and serum starved overnight. Cells were wounded and left untreated or stimulated with 1 nmol/L EGF for 48 h. Images were taken at 0, 24, and 48 h and the migration distances were quantified as in B. Representative of two experiments. F. Western blot analysis showing EphA2 protein levels in HN5 cell lysates prepared from the cells used in the motility assay shown in E. B, D, and E. Bars, SE. *, P < 0.05; **, P < 0.001; ***, P < 0.0001, compared with the zero time point.

Alternatively, it could indicate involvement of other signaling pathways; however, further studies using a panel of molecular inhibitors targeting signaling pathways downstream of ligand- activated EGFR showed no effect on the level of EphA2. A luciferase reporter assay was done to investigate if the increase in EphA2 expression was through a direct effect on the EphA2 promoter. EGF stimulation of the transfected cells resulted in a significant increase in luciferase activity, indicating that the increase in EphA2 levels mediated by EGFR is through activation of the EphA2 promoter. EphA2 is frequently overexpressed in human cancers, and our results suggest that activated EGFR and EGFRvIII could contribute to this through activation of the MEK signaling pathway and induction of EphA2 promoter activation.

EphA2 Localizes with EGFR at the Cell Membrane E-Cadherin has been reported to affect the subcellular localization of EphA2 by directing EphA2 into cell-cell contact (38). In this article, the subcellular localization of EphA2 was investigated in EGFR-expressing human cancer cells by confocal microscopy. In A431 cells, EphA2 was mainly located at cell-cell contacts, whereas in the HN5 cell line, EphA2 was more evenly distributed throughout the cells. The fact that A431 cells express high levels of E-cadherin whereas HN5 cells are devoid of E-cadherin and that E- cadherin has been shown to direct EphA2 into cell-cell contacts (38-40) may explain the difference between the observed distributions of EphA2. Some EphA2 staining was occasionally seen concentrated in the nucleus. It is established that the intracellular fragment of another receptor tyrosine kinase, ErbB4, can be transported to the nucleus. However, whether full-length receptor tyrosine kinases can be transported to the nucleus is controversial (41). The possible function of EphA2 in the nucleus needs to be investigated further. EphA2 showed an overlapping distribu- FIGURE 6. A schematic presentation of two proposed mechanisms based on the presented results. A. EGF stimulation of EGFR induces tion with EGFR at the cell membrane in both HN5 and A431 EphA2 gene transcription. EphA2 and EGFR colocalize and interact on cells. The colocalization was less pronounced in EGF- the plasma membrane of the cancer cells, where ligand stimulation stimulated cells due to EGF-induced EGFR down-regulation. (ephrin-A1) of EphA2 leads to inhibition of EGF-induced cancer cell motility. B. EGF stimulation of EGFR induces EphA2 gene transcription. The colocalization of EGFR and EphA2 led us to investigate a EphA2 and EGFR colocalize and interact on the plasma membrane of the possible association between EGFR and EphA2. Immunopre- cancer cells, where EphA2 has ligand-independent effects on EGF- induced cancer cell motility. Ligand (ephrin-A1) stimulation of EphA2 leads cipitation of EGFR pulled down EphA2 in EGF-stimulated to receptor internalization, down-regulation, and termination of ligand- cells and cells expressing EGFRvIII, suggesting that EphA2 independent effects on cell motility. associates with activated EGFR. The observation that EGFRvIII associates with EphA2 hints that the association including two human cancer cells lines (A431 and HN5). occurs intracellularly because EGFRvIII lacks extracellular Similarly, the cancer-specific constitutively active variant domain I and two thirds of domain II (42). However, it EGFRvIII was found to increase EphA2 mRNA and protein remains to be elucidated whether EphA2 interacts directly or levels in a mouse fibroblast cell line stably transfected with indirectly with EGFR. We investigated if association between EGFRvIII (NR6M). The results were supported by confocal EGFR and EphA2 could result in receptor transphosphoryla- microscopy, which showed that EGF stimulation significantly tion of ligand stimulation. After 15 min of ligand stimulation, increased the staining intensity of EphA2 in both A431 and neither EphA2 nor EGFR showed detectable effects on HN5 cells. phosphorylation of the other receptor. However, further The increased EphA2 expression was dependent on EGFR experiments showed that EphA2 transphosphorylation could tyrosine kinase activity and partially on MEK activity. These be detected after a longer period (24h) of EGF stimulation. findings are in agreement with recent reports showing increased Similarly, receptor association was detected after 24h and not EphA2 levels in RAS-transformed cells (34, 35, 37). The partial after 15 min of EGF stimulation; therefore, it is possible that effect of the MEK inhibitors in two of the three EGFR expressive association of EphA2 and EGFR results in transphos- cell lines could be due to incomplete inhibition in these cell lines. phorylation. In addition, we investigated cross-talk at the

level of ERK because ligand stimulation of EphA2 has been In summary, our results show that EGFR is an important shown to affect ERK phosphorylation (35, 43). Although inducer of EphA2 expression and that this mechanism may ligand stimulation of EphA2 inhibited basal ERK phosphor- contribute to the high frequency of EphA2 expression found in ylation, no effect on EGF-induced ERK phosphorylation was many human cancers of different origin (26). Furthermore, our detected (not shown). results suggest that EphA2 may play an important role in EGFR-mediated cell motility. Therefore, these results support EphA2 Functions by Modulating EGF-Induced Cell the further development of novel anticancer therapeutics Motility targeting EphA2. The ephrin-EphA2 ligand system plays an important role in repulsion and attraction of neurons and endothelial cells during development (20, 44). In cancer, EphA2 overexpres- Materials and Methods sion has been correlated with metastasis (45, 46), indicating Materials that EphA2 might be involved in cancer cell motility. EGFR is Anti-EphA2 and anti–glyceraldehyde-3-phosphate dehydro- known to induce cell migration (3), and we therefore genase antibodies were purchased from Santa Cruz Biotech- speculated if EphA2 could be involved in regulating EGF- nology (Santa Cruz, CA); polyclonal antibodies to EGFR were induced cancer cell motility. from Fitzgerald Industries (Concord, MA); monoclonal anti- The results show that ligand stimulation of EphA2 signifi- bodies to EGFR (Ab-1) were from Calbiochem (San Diego, cantly inhibits EGF-induced cell migration in a wound healing CA); monoclonal anti-EGFRvIII antibodies were from Novo- assay and in a three-dimensional motility assay (Fig. 5A-D). In castra (Newcastle, United Kingdom); antibodies against correlation, Guo et al. (47) recently reported that EphA2 has phosphorylated and total p44/p42 ERK were from Cell tumor suppressive functions in mammalian skin and suggested Signaling Technology (Danvers, MA); antiphosphotyrosine that EphA2 overexpression in tumors is the result of a (PY-20) monoclonal antibodies were from Zymed Laboratories compensatory feedback defensive mechanism against malignant (San Francisco, CA); and antibody to tubulin was from transformation. In addition, Pedersen et al. have shown that NeoMarkers (Fremont, CA). activated EGFR induces transcription of a panel of genes (IFN-g Recombinant human EGF, AG1478, U73122, PP2, and responsive genes) whose products have known inhibitory effects SU6656 were purchased from Calbiochem. LY294002 was on EGFR activity and function, indicative of a negative feedback purchased from Biosource (Camarillo, CA). U0126 and mechanism abrogating or limiting the tumor-promoting effects of PD98059 were purchased from Promega (Mannheim, Ger- overactivated EGFR (9, 48). Furthermore, Macrae et al. (35) many). SB203580 and wortmannin were purchased from recently showed that RAF activation stimulates EphA2 expres- Upstate Cell Signaling Solution (Lake Placid, NY). Ephrin- sion and that ligand-stimulated EphA2 attenuates activation of A1/Fc was purchased from R&D Systems Europe Ltd. the mitogen-activated protein kinase pathway thereby forming a (Abingdon, United Kingdom). Gefitinib was kindly provided negative feedback loop. Moreover, they reported that expres- by AstraZeneca A/S (Albertslund, Denmark). sions of EphA2 and its ligands are mutually exclusive in breast cancer cell lines and suggested that the interaction between Cell Culture EphA2 and its ligands occurs between cells of different types. The cell lines NR6, NR6wtEGFR, and NR6M have Based on these results, Macrae et al. (35) proposed that ephrin previously been described (9). The human epidermoid ligands may suppress neighboring EphA2-expressing tumor carcinoma cell line A431 was obtained from the American cells depending on RAS signaling and that escape from this Type Culture Collection and the head and neck carcinoma cell inhibition could be a necessary step in development of some line HN5 was kindly provided by Dr. Jiri Bartek (Department of cancers. In conjunction, these observations support the proposed Cell Cycle and Cancer, Danish Cancer Society, Copenhagen, tumor suppressive function of the ephrin-EphA2 ligand system Denmark). All cells were maintained in DMEM (Invitrogen, and is depicted in Fig. 6A. However, EphA2 is overexpressed in Taastrup, Denmark) supplemented with 10% FCS, 50 units/mL a broad range of cancers, and results have shown that EphA2 penicillin, and 50 Ag/mL streptomycin. overexpression is able to induce cancer cell transformation (37). The results from this study show that EphA2 knockdown by Immunoblot Analyses siRNA inhibits EGF-induced cancer cell migration in a wound For the immunoblot analyses, cells were seeded in DMEM with healing assay. These results suggest that EphA2 has a ligand- 10% FCS, allowed to grow for 24h until 80% confluent, washed independent stimulatory effect on EGF-induced cancer cell once in PBS, and serum starved in DMEM supplemented with motility and that EphA2 down-regulation results in termination 0.5% FCS for 48 h. Serum-starved cells were treated with different of these effects (Fig. 6B). In support, the results from the confocal inhibitors using the following concentrations: 5 Amol/L AG1478, microscopy analysis showed that EphA2 ligand stimulation 1 Amol/L gefitinib, 5 Amol/L LY294002, 50 Amol/L PD98059, resulted in removal of EphA2 from the plasma membrane. 10 Amol/L PP2, 10 Amol/L SB203580, 15 Amol/L SU6656, Ligand-induced down-regulation of EphA2 is reported in other 20 Amol/L U0126, 100 nmol/L U73122, and 0.1 Amol/L wort- studies (49, 50). Furthermore, unlike most other receptor tyrosine mannin. Immunoblot analyses were done as previously described kinases, results have shown that EphA2 is constitutively active (9). Briefly, 5 Agofwhole-celllysatewereresolvedbySDS- and does not require ligand-based activation and phosphoryla- PAGE and electroblotted onto nitrocellulose membranes. After tion for enzyme activity (15, 51). Rather, ligand activation seems transfer and blocking in 5% nonfat milk, incubation with to control EphA2 turnover in the plasma membrane. primary antibody was done overnight at 4jC and secondary

antibody staining was done for 1 h at room temperature. The Assay System (Promega) as previously described (52). Differ- enhanced chemiluminescence detection method was used for all ences in luciferase activity were tested using two-sided t test for Western blot experiments. Band intensities were determined using comparing means. the public domain Java image-processing program ImageJ and analyzed using GraphPad Prism with nonlinear regression. Immunofluorescence Confocal Microscopy Serum-starved A431 and HN5 cells plated on CC2-coated Quantitative Real-time Reverse Transcription-PCR chamber slides (Lab-Tek; Nalgene Nunc International, Roches- A Total RNA was extracted from the cells using Trizol reagent ter, NY) were incubated with 10 nmol/L EGF, 1 g/mL ephrin- (Invitrogen, Taastrup, Denmark). The first-strand cDNA syn- A1/Fc (secreted forms of the EphA2 ligand ephrin-A1 attached thesis and the real-time reverse transcription-PCR reaction were to Fc portion of human immunoglobulin G1 heavy chain), or carried out using the SuperScript III Platinum Two-Step qRT- left unstimulated for 48 h. Cells were fixated in 2% PCR Kit with SYBR Green (Invitrogen) with the following paraformaldehyde for 20 min. Nonspecific binding was blocked EphA2 primer sets: EPHA2F, 5¶-CACCTCTGCCAGC- and cells were permeabilized with 5% goat serum (Dako, GACGTG-3¶; EPHA2R, 5¶-CCGCCATGAAGTGCTCCGTA- Glostrup, Denmark) and 0.2% saponin in PBS (blocking buffer) 3¶ (TAG Copenhagen, Denmark). The housekeeping primers for 20 min followed by incubation with anti-EphA2 rabbit (RPL13A-R and RPL13A-F) were from DNA Technology antibody (Santa Cruz Biotechnology) in blocking buffer for 1 h, (Aarhus, Denmark). The PCR cycling conditions used were as wash with PBS, and incubation with antirabbit antibody follows: 95jC for 10 min, 95jC for 20 s, 56.7jC for 20 s, and conjugated to Alexa Fluor 488 (Molecular Probes, Eugene, 72jC for 20 s for 40 cycles. The EphA2 PCR products were OR). After washing with PBS and incubation with blocking normalized to RPL13A PCR products, the level of EphA2 buffer for 20 min, cells were incubated with anti-EGFR mouse mRNA was determined, and the relative amounts of EphA2 antibody (Ab-1; Calbiochem) followed by wash with PBS and mRNA were calculated from the standard curve obtained using incubation with antibody conjugated to Alexa Fluor 568 and known amounts of cDNA. The experiment was repeated twice To-Pro-3 (Molecular Probes). Cells were washed with PBS and and each sample was assayed in duplicates. mounted with Fluoromount G (Southern Biotechnology Associates, Birmingham, AL). Images were acquired with a LSM 510 Meta confocal microscope (Carl Zeiss, Jena, Immunoprecipitation Germany) with 20Â or 40Â objectives and processed using Cell lysates were precleared and equal amounts of protein the LSM software v. 3.2 (Carl Zeiss) and Adobe Photoshop 7.0. (1,000 Ag) were immunoprecipitated with 5 Ag of mouse anti- Images of randomly selected areas from each sample were EGFR (Ab-1; Calbiochem) or 5 Ag of mouse antiphosphotyr- acquired with one of two settings of scanning parameters (low osine (PY-20; Zymed Laboratories). The immunocomplexes magnification: 20Â objective with 3-Am confocal slide, or high were precipitated with protein G agarose beads (Upstate Cell magnification: 40Â objective with 1-Am confocal slide). Using Signaling Solution), collected by centrifugation, and washed the LSM software v. 3.2, the mean intensity of EphA2 and thrice with ice-cold PBS. Antibody control was prepared by EGFR staining was calculated for both settings. Background incubating 1 mL of precleared radioimmunoprecipitation assay pixels were discarded in the mean intensity calculation by buffer with 5 Ag of mouse anti-EGFR antibody followed by setting a threshold corresponding to intensity in a background incubation with antibody and then protein G agarose beads. area with no cells. Difference between images of unstimulated Immunoprecipitated/coimmunoprecipitated proteins were ana- and stimulated cells was tested using two-sided t test for lyzed by Western blot analyses. comparing means. Five to 10 images with 41 to 386 cells from each setting were used. Luciferase Reporter Assay The plasmid À4,030-EphA2-Luc contains a human EphA2 promoter fragment spanning nucleotides À4,030 to +21 Wound Healing Assay relative to the putative EphA2 transcriptional start site in HN5 cells were seeded in triplicate in fibronectin-coated six- front of a luciferase reporter gene and was kindly provided by well plates or T25 cell culture flasks and allowed to grow until David M. Cohen (Oregon Health and Science University and 70% confluence. Cells seeded in T25 flasks were transfected the Portland Veterans Affairs Medical Center, Portland, OR; using 2 AL of LipofectAMINE 2000 reagent (Invitrogen), ref. 36). 4,000 AL of OptiMEM I Reduced Serum Medium (Invitrogen), A431 and HN5 cells, seeded in 24-well plates (100,000 per and 25 nmol/L siRNA targeting EphA2 (Hs_EphA2_8 and well) in DMEM supplemented with 10% FCS and cultured Hs_EphA2_6, Qiagen, Ballerup, Denmark), 25 nmol/L stealth overnight, were transiently transfected with the À4,030-EphA2- RNAi negative control (Invitrogen), or 25 nmol/L BlOCK-iT Luc plasmid or with a control plasmid containing the SV40 Fluorescent Oligo siRNA (Invitrogen). Transfection complex promoter using LipofectAMINE 2000 reagent (Invitrogen). was removed from the cells after 4h and the cells were grown Twenty-four hours after the transfection, cells were switched to overnight in low-serum media. Fluorescence was detected to low-serum medium (0.5%). After 2 h of starvation, cells were analyze transfection efficiency and EphA2 protein levels were stimulated with 5 nmol/L EGF alone or in combinations with 5 detected by Western blot analysis to verify EphA2 knockdown. Amol/L AG1478 or 1 Amol/L gefitinib for 6 h. Cells were then The cell layer was gently ‘‘wounded’’ through the central axis lysed by adding 100 AL/well of Passive Lysis Buffer (Promega) of the plate using a pipette tip. Loose cells were washed off and and luciferase activity was measured using the Luciferase the remaining cells treated with 1 nmol/L EGF and/or 2 Ag/mL

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Mol Cancer Res 2007;5(3). March 2007 Downloaded from mcr.aacrjournals.org on September 29, 2021. © 2007 American Association for Cancer Research. Activation of the EGFR Gene Target EphA2 Inhibits Epidermal Growth Factor−Induced Cancer Cell Motility

Alice Bjerregaard Larsen, Mikkel Wandahl Pedersen, Marie-Thérése Stockhausen, et al.

Mol Cancer Res 2007;5:283-293.

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