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. Author manuscript; available in PMC: 2009 Aug 1.
Published in final edited form as: Clin Cancer Res. 2008 Aug 1;14(15):4943–4950. doi: 10.1158/1078-0432.CCR-08-0436

hMena+11a Isoform Serves as a Marker of Epithelial Phenotype and Sensitivity to EGFR Inhibition in Human Pancreatic Cancer Cell Lines

Maria S Pino 1, Michele Balsamo 2,7, Francesca Di Modugno 3, Marcella Mottolese 4, Massimo Alessio 5, Elisa Melucci 4, Michele Milella 1, David J McConkey 6, Ulrike Philippar 7, Frank B Gertler 7, Pier Giorgio Natali 2, Paola Nistico 2
PMCID: PMC2586967  NIHMSID: NIHMS73685  PMID: 18676769

Abstract

Purpose

hMena, member of the Ena/VASP protein family, is a cytoskeletal protein that is involved in the regulation of cell motility and adhesion. The aim of this study was to determine whether or not the expression of hMena isoforms correlated with sensitivity to EGFR tyrosine kinase inhibitors and could serve as markers with potential clinical use.

Experimental design

Human pancreatic ductal adenocarcinoma (PDAC) cell lines were characterized for in vitro sensitivity to erlotinib, expression of HER family receptors, markers of epithelial to mesenchymal transition (EMT), and expression of hMena and its isoform hMena+11a. The effects of EGF and erlotinib on hMena expression as well as the effect of hMena knock-down on cell proliferation were also evaluated.

Results

hMena was detected in all of the pancreatic tumor cell lines tested as well as in the majority of the human tumor samples [primary (92%) and metastatic (86%)]. Intriguingly, in vitro hMena+11a isoform was specifically associated with an epithelial phenotype, EGFR dependency and sensitivity to erlotinib. In epithelial BxPC3 cells EGF upregulated hMena/hMena+11a and erlotinib downregulated expression. hMena knock-down reduced cell proliferation and MAPK and AKT activation in BxPC3 cells and promoted the growth inhibitory effects of erlotinib.

Conclusions

Collectively, our data indicate that the hMena+11a isoform is associated with an epithelial phenotype and identifies EGFR dependent cell lines that are sensitive to the EGFR inhibitor erlotinib. The availability of anti-hMena+11a specific probes may offer a new tool in pancreatic cancer management if these results can be verified prospectively in cancer patients.

Keywords: hMena, Erlotinib, Sensitivity

INTRODUCTION

Cancer of the exocrine pancreas is the fourth leading cause of cancer related-death worldwide (1). Our inability to detect pancreatic cancer at an early stage, the inherent aggressiveness of the disease, and the lack of effective systemic therapies are responsible for nearly identical incidence and mortality rates. Chemotherapy with gemcitabine has represented the mainstay and the only treatment for patients with advanced pancreatic cancer for the past several decades, providing clinical benefit response with a small improvement in survival (2). As a result and because of a better knowledge of the signaling pathways involved in pancreatic tumorigenesis, alternative treatments combining gemcitabine with other chemotherapeutic or molecularly targeted agents have been proposed (3-9).

Amplification and/or overexpression of the epidermal growth factor receptor (EGFR) and its ligands have frequently been observed in a variety of human malignancies including pancreatic cancer in which the receptor is overexpressed in about 60% of the cases being associated with disease progression and resistance to conventional therapy (10-18). In view of this blockade of EGFR-mediated signal transduction should result in broad suppression of tumor growth (13, 16-19). Indeed erlotinib (TarcevaTM; Roche, Basel, Switzerland and OSI, Melville, NY), an orally bioavailable small molecule inhibitor of the EGFR tyrosine kinase domain, recently became the first targeted therapy clinically approved, in combination with gemcitabine, for the treatment of locally advanced or metastatic pancreatic cancer (5). However, although statistically significant, the improvement in survival was small, calling for better prospective selection of the patients who might benefit based on the identification of reliable predictive molecular marker(s) that identify EGFR-dependent tumors (20-22). The role of EGFR mutation as a principal mechanism in conferring sensitivity to EGFR inhibitors has become controversial and moreover, mutations predictive of outcome have not been found in pancreatic cancer (23-25). Mechanisms aside from mutation of the EGFR tyrosine kinase domain must therefore dictate drug sensitivity.

Increasing evidence suggests that during tumor progression, malignant cells exploit critical developmental and tissue remodeling programs, often promoting a plastic phenotype referred to as an epithelial to mesenchymal transition (EMT). This process, facilitated by the soluble factors continuously exchanged between the neoplastic and the microenvironment-associated cells, is characterized by a loss of E-cadherin expression and concurrent expression of mesenchymal markers such as fibronectin, vimentin, and N-cadherin. These events lead to a subsequent disassembly of cell adherens junctions and the acquisition of a more motile and invasive phenotype through a significant reorganization of the actin cytoskeleton (26-29). Of interest, a recent gene profiling study showed that in non-small cell lung cancer an EMT-like transition is predictive of erlotinib resistance in vitro and in vivo (22). Furthermore Buck et al. reported that pancreatic and colorectal tumor cell lines that expressed low levels or mutant forms of the epithelial junctions constituents E-cadherin and γ-catenin and gained expression of mesenchymal proteins such as vimentin, zeb1 and snail were more resistant to EGFR inhibition in vitro. Gain of mesenchymal markers in human pancreatic and colorectal tumor tissues was also associated with a more advanced tumor stage (30). More recently, Black et al. showed that in a panel of urothelial carcinoma cell lines loss of E-cadherin expression and enhanced invasive/tumorigenic potential were markers of poor response to the antiproliferative effect of EGFR targeting by the monoclonal antibody cetuximab (31). Similar results were also reported previously with gefitinib (32).

Enabled/vasodilator-stimulated phosphoprotein (Ena/VASP) are key regulatory molecules controlling cell shape, movement and actin organization at cadherin adhesion contacts, which are frequently affected following malignant transformation (33). We have shown that human Mena (hMena), a member of the Ena/VASP family, is overexpressed in human breast tumors, and a splice variant termed hMena+11a was recently isolated from a breast cancer cell line with an epithelial phenotype. Of interest, experimental data suggest that hMena couples tyrosine kinase signaling to the actin cytoskeleton (34-36).

The overall goal of our research is to identify molecular markers associated with EGFR dependency that could help clinicians to prospectively select pancreatic cancer patients who are most likely to benefit from erlotinib and other EGFR antagonists. Here we show that the hMena+11a isoform is a marker of epithelial phenotype and EGFR-activation, prerequisite to the antiproliferative effect of EGFR inhibition in human pancreatic cancer cells.

MATERIALS AND METHODS

Reagents

Erlotinib was kindly provided by Roche. A stock solution was prepared in DMSO and stored at –20°C. Recombinant human EGF was purchased from Promega (Madison, WI). The antibodies used for Western blot analyses were from the following sources: rabbit anti-total AKT, rabbit anti-pAKT (Ser473), rabbit anti-total MAPK (MAPK, p42/44), and mouse anti-pMAPK (Thr202-Tyr204) were from Cell Signaling Technology (Beverly, MA); rabbit anti-pEGFR (Tyr1068) was from Biosource (Camarillo, CA); rabbit anti-total EGFR, rabbit anti-HER2, mouse anti-HER3 and rabbit anti-HER4 were from Santa Cruz; mouse anti-E-Cadherin from BD Biosciences; mouse anti-N-Cadherin and mouse anti-vimentin from DakoCytomation (Glostrup, Denmark), and mouse anti-βactin was from Sigma-Aldrich (Poole, United Kingdom). We previously developed the anti-Mena rabbit polyclonal antibody (CKLK1) against the 20 amino acid C-ter peptide of hMena. The anti-Mena+11a rabbit polyclonal antibody was developed against the first 20 amino acid of the human 11a exon (RDSPRKNQIVFDNRSYDSLH) sequence (Covance Research Products, Denver, PA). The human 11a peptide with a terminal cysteine residue was covalently bound to an Iodoacetyl-based resin (SulfoLink Coupling Gel, Pierce, Rockford, IL) and the antibodies were affinity purified following manufacturer’s instruction. The specificity of the antibody was evaluated on Ena/VASP deficient cells stably expressing EGFP-Mena or EGFPMena+11a.

Cell lines and culture conditions

The following cell lines were purchased from the American Type Culture Collection (Rockville, MD): BxPC3, Panc1, MiaPaCa-2, Hs766T and HEK 293 Phoenix (human embryonic kidney cells). The L3.6pl human pancreatic cancer cell line was kindly provided by Dr I.J. Fidler (The University of Texas M.D. Anderson Cancer Center, Houston, TX). The T3M4, PACA44, and PT45 cell lines were kindly provided by Dr. F. Velotti (Tuscia University, Viterbo, Italy). All cell lines were maintained in RPMI 1640 (Life Technologies, Inc., Gaithersburg, MD). The medium was supplemented with 10% fetal bovine serum (FBS; Life Technologies), L-glutamine (Bio Whittaker, Rockland, ME), and antibiotics (penicillin/streptomycin; Bio Whittaker). Adherent monolayer cultures were incubated at 37°C in a mixture of 5% CO2 and 95% air. MVD7, Ena/VASP deficient mouse embryonic fibroblastic cells, were isolated as described in Bear et al. and cultured at 32°C in Immorto media [high-glucose Dulbecco’s modified Eagle’s with 15% fetal calf serum, penicillin/streptomycin, l-glutamine, and 50 U/ml recombinant mouse interferon-γ (Invitrogen, Carlsbad, CA)] (37).

Retroviral Packaging, Infection and FACS Sorting

EGFP-Mena and EGFP-Mena+11a were subcloned into pMSCV-EGFP retroviral plasmid by using standard techniques. Retroviral plasmids were transiently transfected into 293 Phoenix cells and supernatant was collected after 48 h. MVD7 cells were exposed to infectious supernatant for 24 hours in the presence of 4 μg/ml polybrene and cultured to 90% confluence, trypsinized, and FACS sorted in phosphate-buffered saline/5% fetal calf serum. EGFP-positive cells were harvested and sorted for EGFP signal intensity levels that matched EGFP-Mena and EGFP-Mena+11a.

Cell treatments

Cells were grown in six-well plates to confluence in RPMI supplemented with 10% fetal bovine serum. After 18 hours in serum-free medium, the cells were treated with different amounts of rhEGF for 24 hours. Erlotinib (10 μmol/L) was added 2 hours before EGF treatment.

Western blot analyses

Cells were lysed as reported (34). Lysates (30 or 100 μg) were resolved on 10% polyacrylamide gel and transferred to nitrocellulose membranes (GE Healthcare, Piscataway, NJ). Membranes were blocked in 5% nonfat milk in Tris-buffered saline (TBS) containing 0.1% Tween 20 (TBS-T) for 1 hour at room temperature, incubated overnight with relevant antibodies, washed, and probed with species-specific secondary antibodies coupled to horseradish peroxidase, and detected by enhanced chemiluminescence (Amersham Biosciences).

Proliferation assays

Cell proliferation was determined by measurement of cellular ATP levels with a high sensitivity cell proliferation/cytotoxicity kit (Vialight Plus, Cambrex Bio Sci Rockland Inc., Rockland, ME). Briefly, cells were plated in 96-well plates at a density of 1 × 104 per well and exposed 24 hours later to various concentrations of erlotinib (0.1-10 μmol/L) in serum-free media. After 24 hours nucleotide (ATP) releasing reagent (100 μl) was added to each well and the plate was incubated for 10 minutes at room temperature. Cell lysate (180 μl) was transferred to a luminescence compatible plate. The 96-well plates were read using a Perkin Elmer LS 50B luminometer. ATP levels in cells were normalized to levels in untreated control cultures.

Small interfering RNA treatment

Cells in exponential growth phase were transfected with 100 nmol/L hMena-specific pooled small interfering RNA (siRNA) duplexes (siENA SMART pool) or control nonspecific siRNA (Dharmacon, Lafayette, CO) using LipofectAMINE 2000 reagent (Invitrogen, Paisley, United Kingdom). After culturing for 48 hours, cells were serum deprived for 18 hours and then differently treated for Western blot analysis or proliferation assays.

Two-dimensional electrophoresis

Cells were washed, lyophilized, and proteins solubilized with two-dimensional electrophoresis buffer [9 mol/L urea, 10 mmol/L Tris, 4% CHAPS, 65 mmol/L DTT, 2% IPG buffer ampholine (pH 3–10), protease inhibitor cocktail]. Protein samples (250 μg) were applied to 7-cm IPG strips pH 3–10 nonlinear (Amersham Biosciences) and isoelectrofocusing was done with an IPGphor system (Amersham Biosciences) following a standard protocol as described (38). Strips were equilibrated in 50 mmol/L Tris-HCl buffer (pH 8.8) containing 6 mol/L urea, 30% glycerol, 2% SDS, and 2% DTT, followed by an incubation in the same buffer replacing DTT with 2.5% iodoacetamide. The strips were loaded on top of 10% acrylamide SDS-PAGE gels for the second dimension separation. Proteins were electrontransferred onto nitrocellulose membranes and Western blot was done as described above. Images were acquired at high resolution and two-dimensional immunoreactivity patterns analyzed using Progenesis PG240 v2005 software (Nonlinear Dynamics, Newcastle, United Kingdom). Relative molecular mass (Mr) was estimated r by comparison with Mr reference markers (Precision, Bio-Rad, Hercules, CA) and isoelectric point (pI) values assigned to detected spots by calibration as described in the Amersham Biosciences guidelines.

Patients and tissue specimens

A series of 26 patients (median age 62 yrs; range 39-78) who underwent pancreatic resection or biopsy at the Regina Elena National Cancer Institute between 2002 and 2005 with a diagnosis of pancreatic adenocarcinoma were retrospectively collected for immunohistochemical studies. This series included 12 primary (9 stage II, 1 stage III, 2 stage IV) and 14 metastatic carcinomas (11 liver metastasis, 3 other abdominal sites). Tumors were staged according to the Unione Internationale Contre le Cancer TNM System 2002 and collected according to the Internal Ethic Committee guidelines.

Immunohistochemistry

Pancreatic cancer specimens were fixed for 18-24 hours in buffered formaldehyde and then processed through to paraffin wax. hMena expression was evaluated by immunohistochemistry using the mAb clone 21 (BD Transduction, San Jose, CA; 2.5 μg/mL) that recognizes all the hMena isoforms and does not cross-react with other members of Ena/VASP family proteins (34). Dewaxing, antigen retrieval, incubation with the primary antibody, chromogenic reaction with 3,3’-diaminobenzidine (DAB) and counterstaining with Mayer Haematoxylin were performed with an automatic autostainer (Vysion Biosystems Bond, Menarini, Florence, Italy). Sections were mounted in aqueous mounting medium (Glycergel, DakoCytomation). The intensity of hMena staining, detected in the cytoplasm, was scored from 0 to 3+ according to the following criteria: no staining, score 0; weak cytoplasmic staining of neoplastic cells, score 1+; moderate cytoplasmic staining, score 2+; strong cytoplasmic staining, score 3+. Evaluation of the immunohistochemical data was done independently and in blinded manner by two investigators.

Statistical analysis

All experiments were repeated at least twice. Statistical significance was determined by Student’s t test (two tailed) comparison between two groups of data. Asterisks indicate significant differences of experimental groups compared with the corresponding control condition (* P < 0.05; ** P < 0.01). Statistical analysis was done using GraphPad Prism 4, V4.03 software (GraphPad, Inc., San Diego, CA). Change in the phosphorylation status was evaluated, using Progenesis v.2004 software (Nonlinear Dynamics), by absorbance indicated as normalized spot volume. Normalization was done by multiplying the total spot volume by the constant factor 100, which produces spot percentage volume. Densitometric quantitation of hMena immunoreactivity was determined by ImageJ and normalized in comparison with the actin immunoreactivity.

RESULTS

hMena and hMena+11a isoform expression in pancreatic cancer-derived cell lines

To acquire insights into the expression, modulation and function of the hMena and its isoform in pancreatic cancer, we first characterized the hMena and hMena+11a expression in a panel of eight pancreatic cancer cell lines by Western blot analysis. Using an anti-hMena antibody recognizing all isoforms (pan-hMena) we observed (Figure 1A) that hMena was consistently expressed at different level in all the tumor cell lines tested. Since hMena and hMena+11a isoforms are not distinguishable by Western blot because they comigrate (88-90 kDa), we used an anti-hMena+11a antibody that specifically recognize this isoform. The specificity of this antibody was tested on cell lisates from Ena/VASP deficient cells, stably expressing EGFP-Mena and EGFP-Mena+11a (Figure 1B). hMena+11a was selectively expressed in four out of the eight cell lines (L3.6pl, BxPC3, T3M4 and PACA44) and in the HPDE normal cell line (data not shown), whereas it was undetectable in PT45, Panc1, MiaPaCa-2 and Hs766T cell lines. Furthermore, a two-dimensional Western blot analysis was conducted on protein extracts from two representative cell lines, BxPC3 and Panc1. In BxPC3 two distinct sets of spots with slightly different molecular mass and pI ranging between 5.4 to 6 (lower protein spots) and 5.8 to 6.2 (upper protein spots) was revealed by pan-hMena (Figure 1C). These two set of spots correspond to the two different isoforms, hMena and hMena+11a as previously reported in breast cancer (36). A different pattern was observed in Panc1 cells, which like BxPC3 cells expressed the 5.4 to 6 pI set of spots (hMena). However, the set of spots corresponding to hMena+11a were absent, and a new set of protein spots displaying a lower molecular weight and more basic pI (range, 5.9–6.7) was present. (We are currently characterizing this isoform in more detail.) Since expression of hMena+11a appears to be restricted to cells with an epithelial phenotype, we evaluated markers of epithelial to mesenchymal transition (EMT) in our panel of pancreatic cancer cell lines by Western blot analysis. As shown in Figure 1D, E-cadherin was highly expressed in all of the hMena+11a positive cell lines (L3.6pl, BxPC3, T3M4 and PACA44) and was absent in the hMena+11a negative cell lines. Conversely, we detected expression of the mesenchymal marker vimentin in PT45, Panc1 and MiaPaCa-2 and N-cadherin in MiaPaCa-2 and Hs766T suggesting that hMena+11a is a marker of an epithelial phenotype in pancreatic cancer cell lines.

Figure 1. hMena and hMena+11a isoform expression in pancreatic cancer-derived cell lines.

Figure 1

Figure 1

Figure 1

Figure 1

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A, protein extracts (25 μg) from pancreatic tumor cell lines were immunoblotted with an anti-hMena CKLK1 antibody and an anti-hMena+11a specific antibody. As loading control the same blots were probed with an anti-actin monoclonal antibody. B, Mena+11a antibody specifically recognizes EGFP-Mena+11a but not EGFP-Mena stably transfected in Ena/VASP deficient cells. C, proteins from two different pancreatic cancer cell lines (BxPC3 and Panc1) were resolved by pI in the first dimension on a pH 3 to 10 nonlinear range and by relative molecular mass in the second dimension on a 10% acrylamide SDS-PAGE. Proteins were then electrotransferred to nitrocellulose and hMena reactivity was revealed by Western blot. Protein spot sets were compared by using Progenesis PG240v2005 software. The range of pI values assigned to detected spots is indicated. Two sets of spots with different molecular mass and pI ranging between 5.4 to 6.0 (lower protein spots) and 5.8 to 6.2 (upper protein spots), indicated as hMena and hMena+11a, respectively, are clearly discriminated. D, the expression of epithelial (E-cadherin) and mesenchymal (N-cadherin and vimentin) proteins in the panel of pancreatic tumor cell lines was evaluated by Western blot analysis. Actin was used as a protein loading control.

hMena+11a isoform expression correlates with sensitivity to EGFR inhibition in pancreatic cancer cell lines

Recently we have shown that in breast cancer hMena may couple tyrosine kinase signaling to the actin cytoskeleton (36). In view of the role of EGFR as a relevant therapeutic target in the treatment of pancreatic cancer patients, we evaluated the growth inhibitory effect of the EGFR tyrosinekinase inhibitor erlotinib in our panel of pancreatic cancer cell lines by exposing them to increasing concentrations (0-10 μmol/L) of the drug. As shown in Figure 2, we observed a significant heterogeneity in drug responsiveness. Considering the average steady-state plasma concentrations in erlotinib treated patients, we divided our panel in sensitive (L3.6pl, BxPC3, T3M4 and PACA44), displaying at least a 50% inhibition of proliferation at concentrations of erlotinib ≤ 1 μmol/L, and resistant (PT45, Panc1, MiaPaCa-2 and Hs766T) cell lines, in which the growth rate was not significantly affected even with an erlotinib concentration of 10 μmol/L (39, 40). Of interest, all the erlotinib-sensitive cell lines expressed hMena+11a indicating that this isoform identifies a specific cell phenotype in which EGFR-tyrosine kinase inhibition significantly affect cell proliferation. No hMena+11a expression was in fact observed in the erlotinib-resistant cell lines. To evaluate whether the expression of other EGFR family members might affect the responsiveness of tumor cells to EGFR kinase inhibitors we analyzed the levels of EGFR family members in our panel of pancreatic cancer cell lines by Western blot (Figure 3). The expression of HER family members was not correlated with hMena and hMena+11a and no correlation was found with erlotinib sensitivity, confirming previous results (41-43). However, a constitutive EGFR phosphorylation was seen exclusively in the sensitive pancreatic cancer cell lines hMena+11a positive, with PACA44 expressing the lowest level of phosphorylation being the least sensitive among them. This observation is consistent with our previous findings that EGFR-mediated signaling networks are “on” in the erlotinib-sensitive cells, driven by availability of the autocrine ligand production (44).

Figure 2. Concentration-dependent effects of erlotinib on cell proliferation.

Figure 2

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The effects of increasing concentrations of erlotinib (0.1 to 10 μmol/L) on cell proliferation were assessed by measuring the intracellular ATP content using the Vialight assay as described in Materials and Methods. Results are expressed as percentage inhibition of ATP incorporation relative to untreated cells. Erlotinib-sensitive cells (gray) and -resistant cells (black). Representative results of at least three separate experiments are shown. In the raw data, the standard error (SE) did not exceed 10%.

Figure 3. Sensitivity to erlotinib does not correlate with HER family members expression in pancreatic cancer cell lines.

Figure 3

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Protein extracts (100 μg) from erlotinib sensitive pancreatic cancer (L3.6pl, BxPC3, T3M4 and PACA44) and resistant (PT45, Panc1, MiaPaCa-2 and Hs766T) cell lines, were analyzed by Western blot analysis using an anti EGFR and phospho EGFR, HER2, HER3 and HER4 antibodies. As loading control the same blots were probed with an anti-actin monoclonal antibody.

Effect of EGF and erlotinib treatment on hMena expression in pancreatic cancer cell lines

To further test our hypothesis that hMena isoforms are along the EGFR-signaling pathway, we explored the effects of EGF and erlotinib treatment on hMena expression in BxPC3, erlotinib-sensitive, and Panc1, erlotinib-resistant cell lines (Figure 4). Twenty-four hours treatment with two different EGF concentrations (50 and 100 ng/ml) clearly increased hMena and hMena+11a protein level as detected by Western blot analysis in both cell lines. Furthermore, the addition of erlotinib to the EGF-treated cell lines downregulated hMena expression only in the hMena+11a positive BxPC3 cell line.

Figure 4. EGF treatment up-regulates whereas erlotinib downregulates hMena expression in pancreatic cancer cell lines.

Figure 4

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BxPC3 and Panc1 pancreatic cancer cell lines untreated, treated with EGF 50 and 100 ng/ml for 24 hours, or treated with EGF for 24 hours after 2 hours incubation with the EGFR tyrosin kinase inhibitor erlotinib (10 μM/L), were evaluated for hMena and hMena+11a expression. As loading control the same blots were probed with an anti-actin monoclonal antibody. Densitometric quantitation of hMena immunoreactivity is shown at the bottom.

hMena knock-down reduces proliferation, AKT and MAPK activation in the erlotinib-sensitive pancreatic cancer cell lines

In view of hMena+11a role on the proliferative activity in breast cancer cells, we transiently knocked down, with high efficiency, hMena expression in hMena+11a positive, erlotinib-sensitive BxPC3 and in hMena+11a negative and erlotinib-resistant Panc1 cell lines via RNA interference (Figure 5A). In parallel, we analyzed constitutive AKT and MAPK phosphorylation levels and found that they were high in both the cell lines, consistent with the general relevance of these pathways in driving proliferation and survival in pancreatic cancer cells. However, whereas AKT and MAPK phosphorylation were strongly reduced in hMena-silenced BxPC3 cells, hMena knock-down did not or slightly affect constitutive AKT and MAPK phosphorylation in Panc1 cells. The effects of hMena knock-down on these pathways were associated with a significant reduction of the baseline growth rate in BxPC3 (45% vs 100% p=0.002) compared with the growth rate of untransfected cells and cells transfected with a control non specific siRNA. In the Panc1 cell line hMena knock-down also reduced proliferation rates but the effects were much less dramatic (78% vs 100%, p=0.01) (Figure 5B). Notably, combined exposure to the hMena siRNA construct (48 hours) plus erlotinib (100 nM, 24 hours) resulted in an additive decrease in proliferation in the BxPC3 cells but did not in the hMena+11a negative Panc1 cells as compared with untransfected cells (Figure 5B).

Figure 5. hMena knock-down reduces proliferation and phosphorylation of AKT and MAPK in the hMena+11a positive, erlotinib-sensitive pancreatic cancer cell lines.

Figure 5

Figure 5

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A, Western blot analysis of BxPC3 and Panc1 pancreatic cancer cell lines after 72 hours transfection with control and hMena specific siRNAs. Silencing of hMena reduces the constitutive AKT and MAPK phosphorylation in BxPC3 cell line whereas no significant changes were observed in Panc1. As a loading control the same blots were probed with a specific anti-actin antibody. B, silencing of hMena reduces the baseline growth rate in BxPC3 compared with the growth rate of untransfected cells and cells transfected with non specific siRNA (45% vs 100%, P<0.01). Not that in the Panc1 cell line hMena knock-down was significantly less efficient in reducing proliferation (78% vs 100%, P<0.05). Furthermore, a dramatic decrease in cell proliferation was observed in the hMena-silenced BxPC3 cells treated with erlotinib at a concentration of 0.1 μM/L for 24 hours compared with the growth rate of untransfected cells. Proliferation assays were conducted 72 h after the siRNA transfection as described in Material and Methods.

hMena expression in pancreatic cancer lesions of 26 patients

To evaluate the in vivo expression of hMena we analyzed by IHC 12 primary pancreatic tumors and 14 synchronous metastases of 26 patients. As shown in Table 1, pan-hMena, although with various intensities, was detected in 11 out of the 12 primary tumors (92%) analyzed. More specifically, 3 cases (25%) showed a 3+, score and 8 cases (66.6%) a 2+ score whereas only one tumor was negative. Among the 14 metastases, 12 (85.7%) presented a 2+/3+ pan-hMena score whereas 2 cases (14.2%) were negative. Representative cases of hMena expression are shown in Figure 6.

Table 1.

hMena protein expression in invasive and metastatic pancreatic carcinoma by immunohistochemistry

hMena score
No cases 0/1+(%) 2+/3+(%)
Primary tumors: 12 1 (8.4) 11 (91.6)
Metastatic tumors: 14 2 (14.2) 12 (85.8)
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Figure 6. hMena expression in primary and metastatic pancreatic carcinomas by immunohistochemistry.

Figure 6

Figure 6

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A, a representative case of a primary pancreatic carcinoma with a cytoplasmic immunoreactivity score of 2+ for pan-hMena. B, a representative case of a liver metastasis from pancreatic carcinoma with a strong cytoplasmic immunoreactivity score of 3+ for pan-hMena. Magnifications, X10 (A), X20 (B).

DISCUSSION

The feeble and heterogeneous clinical responses to EGFR-inhibitors therapies in patients with locally advanced or metastatic carcinomas highlight the importance of identifying the molecular determinants of patients’ responsiveness which may be translatable to the clinical setting (5). We have previously shown that a subset of gefitinib-sensitive pancreatic cancer cells is dependant on autocrine TGF-α-mediated activation of the EGFR for cell proliferation and display constitutive EGFR phosphorylation in vitro and in vivo, whereas insensitive cell lines do not exhibit TGF-α expression and EGFR constitutive activation (44). To contribute in the understanding of the mechanisms leading to EGFR-inhibitors sensitivity and in light of accumulating data implicating the EGFR in the modulation of the actin cytoskeleton, in the present study we evaluated whether hMena and hMena+11a, actin-binding proteins that are able to couple tyrosine kinase signaling to the actin cytoskeleton in breast cancer, may represent key mediators of EGFR activity in pancreatic cancer cells (36, 45, 46).

The expression of hMena and hMena+11a was first characterized in a panel of human pancreatic cancer cell lines showing heterogeneity in responsiveness to the TKI inhibitor erlotinib. Whereas in other normal tissues hMena expression has been reported at low or no detectable levels, hMena was detected in all the pancreatic tumor cell lines tested, in a human pancreatic ductal epithelial cell line (HPDE), as well as in pancreatic tissue, primary and metastatic tumors (34-36). Intriguingly, the expression of hMena+11a, an isoform specific to cell lines that display an epithelial phenotype, was restricted to the non-neoplastic HPDE cell line and to the four cancer cell lines that were E-cadherin positive and negative for expression of vimentin and N-cadherin. Notably, these four cell lines also displayed constitutive phosphorylation of the EGFR pathway and significant sensitivity to erlotinib suggesting, in agreement with recent published data, that the efficacy of EGFR inhibitors is tightly related to the activation of the targeted pathway (47). Collectively these data lead us to hypothesize that hMena+11a is not only a marker of an epithelial phenotype, but its expression is also able to identify cancer cells that are utilizing the EGFR to drive proliferation, rendering them sensitive to EGFR-specific TKIs. On the contrary, the hMena+11a isoform was not expressed in mesenchymal, erlotinib-resistant cancer cell lines.

Our results are consistent with previous studies that have linked EMT to erlotinib resistance in NSCLC cells in vivo and in vitro (22). Furthermore, in a small subset of patients enrolled in a randomized, placebo-controlled NSCLC clinical trial, in which erlotinib in combination with chemotherapy failed to show clinical activity, a positive E-cadherin immunostaining was predictor of a better outcome in all measures of clinical benefit in patients treated with erlotinib (48, 49). In pancreatic and colorectal tumor cell lines the loss of E-cadherin and often the gain of proteins associated with an EMT phenotype correlates with resistance to EGFR inhibition. Furthermore when the expression of E-cadherin and vimentin was measured in pancreatic and colorectal tissue microarrays containing tumors of varying stages, decreased E-cadherin expression and parallel increased vimentin expression is observed with advanced tumor stages (30). The extent to which the mesenchymal proteins are cellular biomarkers rather than functional participants in producing insensitivity to EGFR inhibitors is unclear at the present time. Recent observations suggest that during EMT cells acquire abnormal EGFR-independent survival signals in part from the activation of either or both the phosphatidyl inositol 3-kinase or Ras-Raf-Mek-Erk pathways (50).

In view of the above, we analyzed the effect of hMena knock-down in BxPC3 and Panc1 cell lines endowed with differential sensitivity to erlotinib. In BxPC3 cells, displaying the “on” EGFR pathway, hMena/hMena+11a knock-down significantly impaired cell proliferation, whereas this effect was barely evident in hMena+11a negative Panc1 cells, possessing an “off” EGFR pathway. This observation was confirmed at the protein level by a significant reduction of the constitutive AKT and MAPK phosphorylation, known to be via EGFR-mediated routes in BxPC3 cell line (44). Conversely, the lack of effect on AKT and MAPK phosphorylation by hMena silencing in Panc1 cells, in which the EGFR pathway is “off”, again support the hypothesis that hMena is downstream to the EGFR activity. As expected, when hMena knock-down cells were treated with very low concentration of erlotinib, a dramatic decrease on cell proliferation was observed only in BxPC3.

In conclusion, we have shown that hMena acts as a mediator of the EGFR signaling pathway and significantly modulates the growth of pancreatic cancer cell lines dependent on EGFR signaling. These cell lines, which display an epithelial phenotype are erlotinib-sensitive and are selectively characterized by the presence of hMena+11a isoform. As a whole, the results of the present study identify the expression of hMena/hMena+11a as predictive of in vitro response to EGFR inhibitors, thus strongly supporting prospective studies to assess whether this molecular signature may be associated with an improved clinical response to EGFR targeted therapy in pancreatic cancer.

Acknowledgments

We thank Dr. Duilia Del Bello and Dr. Angela Santoni for their continuous and precious support; Dr. Franco Novelli for helpful discussion.

Financial support: Lega Italiana per la Lotta Contro i Tumori and Associazione Italiana per la Ricerca sul Cancro (AIRC) (P.N.); AIRC (M.M.1); Fondazione CARIPLO (Nobel GUARD Project, and 2006.0537/105411 Project) (MA); Funding from the Ludwig Center (1U54 CA112967-03) and NIH grant (GM58801) (F.B.G); U.P. was supported by a Ludwig Center Fellowship.

Footnotes

Statement of Clinical Relevance The feeble and heterogeneous clinical responses to EGFR-inhibitors therapies in patients with locally advanced or metastatic pancreatic carcinomas highlight the importance of identifying the molecular determinants of patients’ responsiveness which may be translatable to the clinical setting. Such as strategy not only could enable clinicians to select the best therapeutic agents for each patient but most importantly could spare them unnecessary treatment with ineffective agents. Profiling a panel of pancreatic cancer cell lines, the present study identify hMena and its epithelial specific isoform hMena+11a, actin-regulatory proteins, as predictive of in vitro response to EGFR inhibitors. hMena+11a isoform was specifically associated with an epithelial phenotype selectively identifying EGFR dependant cell lines that are sensitive to the inhibition of this signaling pathway. As a whole our results strongly support prospective studies, in the context of clinical trials, to validate hMena/hMena+11a as markers of response to EGFR targeted therapy in pancreatic cancer patients.

References

  • 1.Jemal A, Siegel R, Ward E, Murray T, Xu J, Thun MJ. Cancer Statistics, 2007. CA Cancer J Clin. 2007;57:43–66. doi: 10.3322/canjclin.57.1.43. [DOI] [PubMed] [Google Scholar]
  • 2.Burris H, 3rd, Moore M, Andersen J, et al. Improvements in survival and clinical benefit with gemcitabine as first- line therapy for patients with advanced pancreas cancer: a randomized trial. J Clin Oncol. 1997;15:2403–13. doi: 10.1200/JCO.1997.15.6.2403. [DOI] [PubMed] [Google Scholar]
  • 3.Heinemann V, Quietzsch D, Gieseler F, et al. Randomized Phase III Trial of Gemcitabine Plus Cisplatin Compared With Gemcitabine Alone in Advanced Pancreatic Cancer. J Clin Oncol. 2006;24:3946–52. doi: 10.1200/JCO.2005.05.1490. [DOI] [PubMed] [Google Scholar]
  • 4.Louvet C, Labianca R, Hammel P, et al. Gemcitabine in Combination With Oxaliplatin Compared With Gemcitabine Alone in Locally Advanced or Metastatic Pancreatic Cancer: Results of a GERCOR and GISCAD Phase III Trial. J Clin Oncol. 2005;23:3509–16. doi: 10.1200/JCO.2005.06.023. [DOI] [PubMed] [Google Scholar]
  • 5.Moore MJ, Goldstein D, Hamm J, et al. Erlotinib Plus Gemcitabine Compared With Gemcitabine Alone in Patients With Advanced Pancreatic Cancer: A Phase III Trial of the National Cancer Institute of Canada Clinical Trials Group. J Clin Oncol. 2007;25:1960–66. doi: 10.1200/JCO.2006.07.9525. [DOI] [PubMed] [Google Scholar]
  • 6.Oettle H, Richards D, Ramanathan RK, et al. A phase III trial of pemetrexed plus gemcitabine versus gemcitabine in patients with unresectable or metastatic pancreatic cancer. Ann Oncol. 2005;16:1639–45. doi: 10.1093/annonc/mdi309. [DOI] [PubMed] [Google Scholar]
  • 7.Lima CM Rocha, Green MR, Rotche R, et al. Irinotecan Plus Gemcitabine Results in No Survival Advantage Compared With Gemcitabine Monotherapy in Patients With Locally Advanced or Metastatic Pancreatic Cancer Despite Increased Tumor Response Rate. J Clin Oncol. 2004;22:3776–83. doi: 10.1200/JCO.2004.12.082. [DOI] [PubMed] [Google Scholar]
  • 8.Van Cutsem E, van de Velde H, Karasek P, et al. Phase III Trial of Gemcitabine Plus Tipifarnib Compared With Gemcitabine Plus Placebo in Advanced Pancreatic Cancer. J Clin Oncol. 2004;22:1430–38. doi: 10.1200/JCO.2004.10.112. [DOI] [PubMed] [Google Scholar]
  • 9.Bria E, Milella M, Gelibter A, et al. Gemcitabine-based combinations for inoperable pancreatic cancer: Have we made real progress? Cancer. 2007;110:525–33. doi: 10.1002/cncr.22809. [DOI] [PubMed] [Google Scholar]
  • 10.Grandis J, Melhem M, Gooding W, et al. Levels of TGF-alpha and EGFR protein in head and neck squamous cell carcinoma and patient survival. J Natl Cancer Inst. 1998;90:824–32. doi: 10.1093/jnci/90.11.824. [DOI] [PubMed] [Google Scholar]
  • 11.Salomon DS, Brandt R, Ciardiello F, Normanno N. Epidermal growth factor-related peptides and their receptors in human malignancies. Crit Rev Oncol Hematol. 1995;19:183–232. doi: 10.1016/1040-8428(94)00144-i. [DOI] [PubMed] [Google Scholar]
  • 12.Kondapaka SB, Fridman R, Reddy KB. Epidermal growth factor and amphiregulin up-regulate matrix metalloproteinase-9 (MMP-9) in human breast cancer cells. Int J Cancer. 1997;70:722–26. doi: 10.1002/(sici)1097-0215(19970317)70:6<722::aid-ijc15>3.0.co;2-b. [DOI] [PubMed] [Google Scholar]
  • 13.Ennis BW, Lippman ME, Dickson RB. The EGF receptor system as a target for antitumor therapy. Cancer Invest. 1991;9:553–62. doi: 10.3109/07357909109018953. [DOI] [PubMed] [Google Scholar]
  • 14.Olayioye MA, Neve RM, Lane HA, Hynes NE. The ErbB signaling network: receptor heterodimerization in development and cancer. EMBO J. 2000;19:3159–67. doi: 10.1093/emboj/19.13.3159. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Yarden Y, Sliwkowski MX. Untangling the ErbB signalling network. Nat Rev Mol Cell Biol. 2001;2:127–37. doi: 10.1038/35052073. [DOI] [PubMed] [Google Scholar]
  • 16.Yamanaka Y, Friess H, Kobrin MS, Buchler M, Beger HG, Korc M. Coexpression of epidermal growth factor receptor and ligands in human pancreatic cancer is associated with enhanced tumor aggressiveness. Anticancer Res. 1993;13:565–9. [PubMed] [Google Scholar]
  • 17.Barton CM, Hall PA, Hughes CM, Gullick WJ, Lemoine NR. Transforming growth factor alpha and epidermal growth factor in human pancreatic cancer. J Pathol. 1991;163:111–6. doi: 10.1002/path.1711630206. [DOI] [PubMed] [Google Scholar]
  • 18.Korc M, Chandrasekar B, Yamanaka Y, Friess H, Buchier M, Beger HG. Overexpression of the epidermal growth factor receptor in human pancreatic cancer is associated with concomitant increases in the levels of epidermal growth factor and transforming growth factor alpha. J Clin Invest. 1992;90:1352–60. doi: 10.1172/JCI116001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Wagner M, Cao T, Lopez ME, et al. Expression of a truncated EGF receptor is associated with inhibition of pancreatic cancer cell growth and enhanced sensitivity to cisplatinum. Int J Cancer. 1996;68:782–7. doi: 10.1002/(SICI)1097-0215(19961211)68:6<782::AID-IJC16>3.0.CO;2-2. [DOI] [PubMed] [Google Scholar]
  • 20.Moasser MM, Basso A, Averbuch SD, Rosen N. The Tyrosine Kinase Inhibitor ZD1839 (“Iressa”) Inhibits HER2-driven Signaling and Suppresses the Growth of HER2-overexpressing Tumor Cells. Cancer Res. 2001;61:7184–8. [PubMed] [Google Scholar]
  • 21.Engelman JA, Janne PA, Mermel C, et al. ErbB-3 mediates phosphoinositide 3-kinase activity in gefitinib-sensitive non-small cell lung cancer cell lines. Proc Natl Acad Sci U S A. 2005;102:3788–93. doi: 10.1073/pnas.0409773102. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Thomson S, Buck E, Petti F, et al. Epithelial to Mesenchymal Transition Is a Determinant of Sensitivity of Non-Small-Cell Lung Carcinoma Cell Lines and Xenografts to Epidermal Growth Factor Receptor Inhibition. Cancer Res. 2005;65:9455–62. doi: 10.1158/0008-5472.CAN-05-1058. [DOI] [PubMed] [Google Scholar]
  • 23.Lynch TJ, Bell DW, Sordella R, et al. Activating Mutations in the Epidermal Growth Factor Receptor Underlying Responsiveness of Non-Small-Cell Lung Cancer to Gefitinib. N Engl J Med. 2004;350:2129–39. doi: 10.1056/NEJMoa040938. [DOI] [PubMed] [Google Scholar]
  • 24.Shepherd FA, Pereira J Rodrigues, Ciuleanu T, et al. Erlotinib in Previously Treated Non-Small-Cell Lung Cancer. N Engl J Med. 2005;353:123–32. doi: 10.1056/NEJMoa050753. [DOI] [PubMed] [Google Scholar]
  • 25.Tsao MS, Sakurada A, Cutz JC, et al. Erlotinib in Lung Cancer-Molecular and Clinical Predictors of Outcome. N Engl J Med. 2005;353:133–44. doi: 10.1056/NEJMoa050736. [DOI] [PubMed] [Google Scholar]
  • 26.Yamaguchi H, Wyckoff J, Condeelis J. Cell migration in tumors. Curr Opin Cell Biol. 2005;17:559–64. doi: 10.1016/j.ceb.2005.08.002. [DOI] [PubMed] [Google Scholar]
  • 27.Karnoub AE, Dash AB, Vo AP, et al. Mesenchymal stem cells within tumour stroma promote breast cancer metastasis. Nature. 2007;449:557–63. doi: 10.1038/nature06188. [DOI] [PubMed] [Google Scholar]
  • 28.Hay ED. The mesenchymal cell, its role in the embryo, and the remarkable signaling mechanisms that create it. Dev Dyn. 2005;233:706–20. doi: 10.1002/dvdy.20345. [DOI] [PubMed] [Google Scholar]
  • 29.Lee JM, Dedhar S, Kalluri R, Thompson EW. The epithelial-mesenchymal transition: new insights in signaling, development, and disease. J Cell Biol. 2006;172:973–81. doi: 10.1083/jcb.200601018. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Buck E, Eyzaguirre A, Barr S, et al. Loss of homotypic cell adhesion by epithelialmesenchymal transition or mutation limits sensitivity to epidermal growth factor receptor inhibition. Mol Cancer Ther. 2007;6:532–41. doi: 10.1158/1535-7163.MCT-06-0462. [DOI] [PubMed] [Google Scholar]
  • 31.Black PC, Brown GA, Inamoto T, et al. Sensitivity to Epidermal Growth Factor Receptor Inhibitor Requires E-Cadherin Expression in Urothelial Carcinoma Cells. Clin Cancer Res. 2008;14:1478–86. doi: 10.1158/1078-0432.CCR-07-1593. [DOI] [PubMed] [Google Scholar]
  • 32.Shrader M, Pino MS, Brown G, et al. Molecular correlates of gefitinib responsiveness in human bladder cancer cells. Mol Cancer Ther. 2007;6:277–85. doi: 10.1158/1535-7163.MCT-06-0513. [DOI] [PubMed] [Google Scholar]
  • 33.Krause M, Dent EW, Bear JE, Loureiro JJ, Gertler FB. ENA/VASP PROTEINS: Regulators of the Actin Cytoskeleton and Cell Migration. Annu Rev Cell Dev Biol. 2003;19:541–64. doi: 10.1146/annurev.cellbio.19.050103.103356. [DOI] [PubMed] [Google Scholar]
  • 34.Di Modugno F, Mottolese M, Di Benedetto A, et al. The cytoskeleton regulatory protein hMena (ENAH) is overexpressed in human benign breast lesions with high risk of transformation and human epidermal growth factor receptor-2-positive/hormonal receptor-negative tumors. Clin Cancer Res. 2006;12:1470–8. doi: 10.1158/1078-0432.CCR-05-2027. [DOI] [PubMed] [Google Scholar]
  • 35.Di Modugno F, Bronzi G, Scanlan MJ, et al. Human Mena protein, a serex-defined antigen overexpressed in breast cancer eliciting both humoral and CD8+ T-cell immune response. Int J Cancer. 2004;109:909–18. doi: 10.1002/ijc.20094. [DOI] [PubMed] [Google Scholar]
  • 36.Di Modugno F, DeMonte L, Balsamo M, et al. Molecular cloning of hMena (ENAH) and its splice variant hMena+11a: epidermal growth factor increases their expression and stimulates hMena+11a phosphorylation in breast cancer cell lines. Cancer Res. 2007;67:2657–65. doi: 10.1158/0008-5472.CAN-06-1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Bear JE, Loureiro JJ, Libova I, Fassler R, Wehland J, Gertler FB. Negative Regulation of Fibroblast Motility by Ena/VASP Proteins. Cell. 2000;101:717–28. doi: 10.1016/s0092-8674(00)80884-3. [DOI] [PubMed] [Google Scholar]
  • 38.Conti A, Ricchiuto P, Iannaccone S, et al. Pigment epithelium-derived factor is differentially expressed in peripheral neuropathies. Proteomics. 2005;5:4558–67. doi: 10.1002/pmic.200402088. [DOI] [PubMed] [Google Scholar]
  • 39.Petty WJ, Dragnev KH, Memoli VA, et al. Epidermal Growth Factor Receptor Tyrosine Kinase Inhibition Represses Cyclin D1 in Aerodigestive Tract Cancers. Clin Cancer Res. 2004;10:7547–54. doi: 10.1158/1078-0432.CCR-04-1169. [DOI] [PubMed] [Google Scholar]
  • 40.Hidalgo M, Siu LL, Nemunaitis J, et al. Phase I and Pharmacologic Study of OSI-774, an Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor, in Patients With Advanced Solid Malignancies. J Clin Oncol. 2001;19:3267–79. doi: 10.1200/JCO.2001.19.13.3267. [DOI] [PubMed] [Google Scholar]
  • 41.Janmaat ML, Kruyt FAE, Rodriguez JA, Giaccone G. Response to Epidermal Growth Factor Receptor Inhibitors in Non-Small Cell Lung Cancer Cells: Limited Antiproliferative Effects and Absence of Apoptosis Associated with Persistent Activity of Extracellular Signal-regulated Kinase or Akt Kinase Pathways. Clin Cancer Res. 2003;9:2316–26. [PubMed] [Google Scholar]
  • 42.Saltz LB, Meropol NJ, Loehrer PJ, Sr, Needle MN, Kopit J, Mayer RJ. Phase II Trial of Cetuximab in Patients With Refractory Colorectal Cancer That Expresses the Epidermal Growth Factor Receptor. J Clin Oncol. 2004;22:1201–8. doi: 10.1200/JCO.2004.10.182. [DOI] [PubMed] [Google Scholar]
  • 43.Campiglio M, Locatelli A, Olgiati C, et al. Inhibition of proliferation and induction of apoptosis in breast cancer cells by the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor ZD1839 (Iressa) is independent of EGFR expression level. J Cell Physiol. 2004;198:259–68. doi: 10.1002/jcp.10411. [DOI] [PubMed] [Google Scholar]
  • 44.Pino MS, Shrader M, Baker CH, et al. Transforming Growth Factor {alpha} Expression Drives Constitutive Epidermal Growth Factor Receptor Pathway Activation and Sensitivity to Gefitinib (Iressa) in Human Pancreatic Cancer Cell Lines. Cancer Res. 2006;66:3802–12. doi: 10.1158/0008-5472.CAN-05-3753. [DOI] [PubMed] [Google Scholar]
  • 45.Katz M, Amit I, Citri A, et al. A reciprocal tensin-3–cten switch mediates EGF-driven mammary cell migration. Nat Cell Biol. 2007;9:961–9. doi: 10.1038/ncb1622. [DOI] [PubMed] [Google Scholar]
  • 46.den Hartigh J, van Bergen en Henegouwen P, Verkleij A, Boonstra J. The EGF receptor is an actin-binding protein. J Cell Biol. 1992;119:349–55. doi: 10.1083/jcb.119.2.349. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 47.Jimeno A, Tan AC, Coffa J, et al. Coordinated Epidermal Growth Factor Receptor Pathway Gene Overexpression Predicts Epidermal Growth Factor Receptor Inhibitor Sensitivity in Pancreatic Cancer. Cancer Res. 2008;68:2841–49. doi: 10.1158/0008-5472.CAN-07-5200. [DOI] [PubMed] [Google Scholar]
  • 48.Yauch RL, Januario T, Eberhard DA, et al. Epithelial versus Mesenchymal Phenotype Determines In vitro Sensitivity and Predicts Clinical Activity of Erlotinib in Lung Cancer Patients. Clin Cancer Res. 2005;11:8686–98. doi: 10.1158/1078-0432.CCR-05-1492. [DOI] [PubMed] [Google Scholar]
  • 49.Herbst RS, Prager D, Hermann R, et al. TRIBUTE: A Phase III Trial of Erlotinib Hydrochloride (OSI-774) Combined With Carboplatin and Paclitaxel Chemotherapy in Advanced Non-Small-Cell Lung Cancer. J Clin Oncol. 2005;23:5892–9. doi: 10.1200/JCO.2005.02.840. [DOI] [PubMed] [Google Scholar]
  • 50.Perrais M, Chen X, Perez-Moreno M, Gumbiner BM. E-Cadherin Homophilic Ligation Inhibits Cell Growth and Epidermal Growth Factor Receptor Signaling Independently of Other Cell Interactions. Mol. Biol Cell. 2007;18:2013–25. doi: 10.1091/mbc.E06-04-0348. [DOI] [PMC free article] [PubMed] [Google Scholar]

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