Cortactin Overexpression Inhibits Ligand-Induced Down-Regulation of the Epidermal Growth Factor Receptor

Research Article

Cortactin Overexpression Inhibits Ligand-Induced Down-regulation of the Epidermal Growth Factor Receptor

Paul Timpson,1 Danielle K. Lynch,1 Daniel Schramek,1 Francesca Walker,2 and Roger J. Daly1

1Cancer Research Program, Garvan Institute of Medical Research, St. Vincent’s Hospital, Sydney, New South Wales and 2Ludwig Institute for Cancer Research, Melbourne Tumour Biology Branch, Royal Melbourne Hospital, Victoria, Australia

Abstract factor receptor signaling, thereby avoiding aberrant cellular Ligand-induced receptor down-regulation by endocytosis is a stimulation (3, 4). In brief, ligand-induced activation of the EGFR critical process regulating the intensity and duration of leads to the assembly of protein signaling complexes that relay receptor tyrosine kinase signaling. Ubiquitylation of specific various biological signals. Concomitantly, recruitment of the receptor tyrosine kinases, for example, the epidermal growth endocytic machinery occurs and subsequently leads to endosomal factor receptor (EGFR) by the E3 ubiquitin ligase c-Cbl, sorting which can result in lysosomal degradation of the receptor provides a sorting signal for lysosomal degradation and leads and termination of the signal or recycling of the receptor back to to termination of receptor signaling. Cortactin, which couples the cell surface (5–8). The relative efficiency of lysosomal the endocytic machinery to dynamic actin networks, is degradation versus recycling determines the potency of EGFR signaling (9). encoded by EMS1, a gene commonly amplified in breast and head and neck cancers. One mechanism whereby cortactin The recruitment of c-Cbl, a ubiquitin ligase that directs activated overexpression contributes to tumor progression is by receptor tyrosine kinases to lysosomal degradation by tagging them enhancing tumor cell invasion and metastasis. However, in with multi-monoubiquitin chains, plays a fundamental role in this study, we show that overexpression of cortactin in HeLa ligand-induced down-regulation of various receptors (10, 11). cells markedly inhibits ligand-induced down-regulation of the Tyrosines 1045 and 1068 in the carboxy terminal region of EGFR. This is independent of alterations in receptor auto- the EGFR are responsible for the direct and indirect recruitment phosphorylation and correlates with impaired c-Cbl phos- of c-Cbl, respectively (12, 13). Subsequently c-Cbl is tyrosine phorylation and association with the EGFR, reduced EGFR phosphorylated, which stimulates its ubiquitin ligase activity (13). ubiquitylation, and sustained EGF-induced extracellular sig- Receptor ubiquitylation via c-Cbl then regulates endocytic trafficking and degradation of the EGFR (5, 6, 14, 15). In addition nal-regulated kinase activation. Furthermore, analysis of a panel of head and neck squamous cell carcinoma (HNSCC) cell to its role as an ubiquitin ligase, c-Cbl functions as an adaptor, lines revealed that cortactin overexpression is associated with binding the scaffolding proteins Cbl-interacting protein of 85 kDa attenuated ligand-induced EGFR down-regulation. Impor- (CIN85) or CD2-associated protein (CD2AP) in a phosphorylation- tantly, RNAi-mediated reduction of cortactin expression in dependent manner (7, 8, 16). In turn, these scaffolds bind an 11q13-amplified HNSCC cell line accelerates EGFR degra- endophilin, which is thought to induce negative curvature of the dation. This represents the first demonstration of modulation plasma membrane. of growth factor receptor signaling by cortactin. Moreover, Importantly, it has recently become evident that the trans- enhanced EGFR signaling due to cortactin overexpression may forming potential of several oncogenic receptor tyrosine kinases is provide an alternative explanation for EMS1 gene amplifica- partly explained by their ability to avoid c-Cbl recruitment and tion in human cancers. (Cancer Res 2005; 65(8): 3273-80) therefore ligand-induced receptor down-regulation (1–3). Various oncogenic versions of the EGFR including the v-erbB protein of the avian erythroblastosis virus and the EGFR mutant EGFRvV, harbor Introduction deletions spanning the c-Cbl docking site (1). Furthermore, a point Control of growth factor receptor signaling is fundamental to a mutation in the direct c-Cbl binding site of the EGFR impairs wide variety of cellular processes including cell proliferation, down-regulation of the receptor and elicits enhanced mitogenic differentiation, motility, and survival. Therefore, growth factor signaling (12). Also, the most oncogenic member of the EGFR receptors are subject to tight regulation in order to induce family, ErbB2/HER2, is poorly coupled to c-Cbl-mediated degra- signaling of an appropriate magnitude for the task required. dation (17). The EGFR can also escape c-Cbl-mediated down- Inappropriate activation and aberrant signaling by particular regulation by its heterodimerization with ErbB2, which impairs its growth factor receptors has been linked with a number of human normal recruitment of c-Cbl and results in both receptor diseases including cancer (1–3). recycling to the cell surface and enhanced, sustained signaling Multiple mechanisms serve to control signaling by the epidermal (18–20). growth factor receptor (EGFR), including the action of protein Cortactin is a multidomain protein consisting of an NH2- tyrosine phosphatases (3). However, a number of studies indicate terminal acidic region, which binds the actin related protein that ligand-induced receptor down-regulation by endocytosis plays (Arp)2/3 complex, a repeat region, which associates with a key role in regulating the propagation and duration of growth filamentous actin, and a COOH-terminal src homology (SH)3 domain, which recruits a variety of cellular proteins (21). Cortactin can stimulate the actin-nucleation activity of the Requests for reprints: Roger J. Daly, Cancer Research Program, Garvan Institute of Arp2/3 complex alone or in combination with N-WASP (22, 23). Medical Research, St. Vincent’s Hospital, Sydney, New South Wales 2010, Australia. Phone: 61-2-9295-8333; Fax: 61-2-9295-8321; E-mail: [email protected]. In addition, cortactin also inhibits debranching and disassembly I2005 American Association for Cancer Research. of dendritic actin networks (24). One cellular role for cortactin is www.aacrjournals.org 3273 Cancer Res 2005; 65: (8). April 15, 2005

Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 2005 American Association for Cancer Research. Cancer Research to bridge the endocytic machinery with components and Growth factor treatment of cells. Cells were starved overnight in regulators of the actin cytoskeleton. For example, cortactin serum-free medium and then simulated for the indicated time period with binds the mechanochemical GTPase dynamin 2, which regulates 100 ng/mL EGF (R&D Systems, Minneapolis, MN). the fission of endocytic vesicles. This interaction has recently Cell lysis, immunoprecipitation, and immunoblotting. Cell lysates were prepared using radioimmunoprecipitation assay buffer (38) containing been shown to participate in receptor-mediated endocytosis (25– 10 Ag/mL aprotinin, 10 Ag/mL leupeptin, 1 mmol/L sodium orthovanadate, 27). Furthermore, a close relative of CIN85, CD2AP, binds to and 1 mmol/L phenylmethylsulfonyl fluoride. Protein concentrations were cortactin in an endocytic complex containing c-Cbl, the EGFR, determined using Protein Assay Reagent (Bio-Rad, Hercules, CA). and endophilin (16). Finally, cortactin and the Arp2/3 complex Immunoprecipitations were done by incubation with the indicated antibody have been shown to colocalize and associate with endosomal for a minimum of 2 hours at 4jC with constant mixing. Protein A- or vesicles, suggesting a role for the actin nucleation activity of G-sepharose [25 Ag, Zymed, San Francisco, CA; prewashed twice with 20 cortactin in vesicle trafficking (28). mmol/L HEPES (pH 7.5)] was then added to the lysate-antibody mixture for Importantly, cortactin is implicated in the progression of certain 1 hour. Immunoprecipitates were then washed thrice with radioimmuno- human cancers. The gene encoding cortactin, EMS1, localizes to precipitation assay buffer and denatured in SDS-PAGE sample buffer by j chromosomal locus 11q13, a region commonly amplified in breast heating for 3 minutes at 100 C. Samples were analyzed by SDS-PAGE, transferred to a polyvinylidene difluoride membrane (Millipore, Bedford, cancers and head and neck squamous cell carcinoma (HNSCC; refs. MA) and subjected to Western blot analysis. Detection of bound antibodies 29, 30). Since cortactin overexpression increases the motility of was by enhanced chemiluminescence (Amersham Biosciences, Pty. Ltd., fibroblasts and endothelial cells (31, 32) and enhances bone Castle Hill, New South Wales, Australia). Densitometry was done using IP metastasis of MDA-MB-231 breast cancer cells in a nude mouse Lab Gel software (Signal Analytics Corp., Vienna, VA). model (33), the explanation to date for EMS1 amplification in Antibodies. Polyclonal EGFR 1005 (for Western blotting), EGFR (1045), human cancers has been that cortactin overexpression promotes CD2AP (H-290) antibodies, and monoclonal EGFR 528 (for immunopreci- tumor cell invasion or metastasis. pitations), EGFR (1068), and growth factor receptor binding protein 2 The overexpression of cortactin in specific cancers, coupled (Grb2) antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). with its involvement in endocytic events and vesicle trafficking, The cortactin monoclonal 4F11 and the Sprouty2 polyclonal antibody were led us to investigate the effects of enhanced cortactin expression purchased from Upstate Biotechnologies, Inc. (Charlottesville, VA). Poly- clonal phospho- and total extracellular signal-regulated kinase (Erk) on EGFR endocytosis. Unexpectedly, cortactin overexpression antibodies (p42/44) were from Cell Signaling Technology (Beverly, MA) inhibited ligand-induced down-regulation of the EGFR, correlating and the monoclonal c-Cbl (for immunoprecipitations) and horseradish with a decrease in c-Cbl phosphorylation and EGFR ubiquityla- peroxidase-conjugated anti-phosphotyrosine RC20 antibodies were from BD tion and sustained downstream signaling. These findings may Transduction Laboratories (Lexington, KY). The polyclonal c-Cbl antibody provide a novel explanation for EMS1 amplification, and hence R2 (for Western blotting) was a kind gift from Dr. Wally Langdon cortactin overexpression, in malignancies in which the EGFR (University of Western Australia) and the monoclonal anti-HA antibody was plays an important role, such as breast and head and neck obtained from Roche Molecular Biochemicals (Castle Hill, New South Wales, cancers. Australia). Internalization and recycling assays. Receptor internalization and recycling assays were done for 40 to 60 hours after transfection as previously Materials and Methods described (39). For the internalization assay, cells were incubated with Tissue culture and transient transfections. HeLa cells were main- [125I]-EGF at 4jC for 1 hour with or without excess unlabeled EGF for the tained in DMEM (Invitrogen Corp., Carlsbad, CA) supplemented with 10% determination of nonspecific binding. After removing unbound ligand, the (v/v) FCS (ThermoTrace Ltd., Melbourne, Victoria, Australia) and 2 mmol/L cells were shifted to 37jC. At each time point, released EGF (cpm in L-glutamine (Invitrogen). The human squamous cell carcinoma cell lines supernatant), surface-bound EGF (acid-sensitive, cell-associated cpm), and FaDu, Detroit 562, SCC9, SCC15, and SCC25 were maintained as previously internalized EGF (acid-resistant, cell associated cpm) were determined. For described (34). Cells were transfected using Polyfect (Qiagene, Pty. Ltd., the recycling assay, cells were incubated with unlabeled EGF for 1 hour at Clifton Hill, Victoria, Australia) following the manufacturers protocol. 4jC. After removing unbound ligand, cells were shifted to 37jC. At each Plasmids. The pRcCMV-cortactin construct and hemagglutinin (HA)- time point, residual bound EGF was removed by acid wash, and surface tagged ubiquitin-encoding plasmid were as described previously (35, 36). receptor levels assessed by binding of [125I]-EGF. Suppression of cortactin expression by RNAi. Nineteen-nucleotide RNAs were chemically synthesized (Ambion, Inc., Austin, TX) based on the sequence 5V-GACUGGUUUUGGAGGCAAAUUUU-3V (27) or 5V-AAGCU- Results GAGGGAGAAUGUCUUGU-3V and 5V-AAGACUGAGAAGCAUGCCUCTCC- 3V (37). Small interfering RNA (siRNA) against lamin A/C was used as a Cortactin overexpression inhibits ligand-induced down- control. For annealing of siRNA, 20 AL of each single-stranded RNA regulation of the epidermal growth factor receptor. Inhibition (50 Amol/L) was incubated with 10 AL5Â annealing buffer [100 mmol/L of cortactin function via expression of the cortactin SH3 domain or potassium acetate, 2 mmol/L magnesium acetate, 30 mmol/L HEPES microinjection of blocking antibodies reduces receptor-mediated (pH 7.4)] for 1 minute at 90jC and then 1 hour at 37jC. The RNA duplexes endocytosis (26). However, the effect of cortactin overexpression, were then stored at À20jC until use. which occurs in certain human cancers, on endocytic receptor 5 One day before transfection, Detroit 562 cells were plated at 1.4 Â 10 down-regulation has not been investigated. In order to address this, cells per well in six-well plates. Each well contained 2 mL of medium. For HeLa cells were transiently transfected with a cortactin expression A A transfection of the cells in one well, 3 L of siRNA duplex (20 mol/L) was construct. The cells were subsequently serum-starved and then diluted into 200 AL OptiMem (Invitrogen) in tube 1. In tube 2, 12 AL stimulated with EGF over a 120-minute time course. EGFR down- OligofectAMINE (Invitrogen) was added to 48 AL OptiMem. Tubes 1 and 2 were incubated for 7 to 10 minutes at room temperature before combining regulation was determined by Western blotting total cell lysates and the solutions. Following incubation for 20 to 25 minutes at room quantitative analysis by densitometry provided a profile of ligand- temperature, 152 AL of OptiMem was added to the mixture. This was then induced receptor down-regulation over time (Fig. 1A and B, added to the cells, which were incubated with the siRNA for 2 to 4 days respectively). In control (vector-transfected) cells, a decrease in prior to starvation and EGF stimulation. total EGFR levels was detectable after 30 minutes of EGF treatment.

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assessed by immunoblotting with an anti-phosphotyrosine antibody. No significant difference in total EGFR tyrosine phosphorylation was detected between control and cortactin-overexpressing HeLa cells (Fig. 3A). Since the specific EGFR tyrosine phosphorylation sites involved in c-Cbl recruitment are tyrosine (Y) 1045, the binding site for the c-Cbl SH2 domain (13), and Y1068, which recruits c-Cbl indirectly via Grb2 (12), we investigated the status of these sites using specific phosphorylation state–specific antibodies. Ligand-induced phosphorylation of both Y1045 and Y1068 was equivalent in control and cortactin-overexpressing cells (data not shown). Also, there was no alteration in c-Cbl/Grb2 association in cortactin-overexpressing cells versus control cells (data not shown). These data indicate that the mechanism responsible for the impaired EGFR down-regulation lies downstream from receptor tyrosine phosphorylation. Overexpression of cortactin impairs coupling of the epidermal growth factor receptor to c-Cbl and epidermal growth factor receptor ubiquitylation. The known role of c-Cbl in both EGFR internalization and down-regulation (3, 4), led us to examine whether this regulatory pathway was altered in cortactin- overexpressing cells. Western blotting of EGFR immunoprecipitates

Figure 1. Overexpression of cortactin inhibits ligand-induced EGFR down-regulation. A, HeLa cells were transiently transfected with a cortactin expression vector (C) or with vector alone (V) and then stimulated with EGF for the indicated time periods. Cell lysates were prepared and Western blotted for the EGFR and cortactin as indicated. A CD2AP blot was also done as a loading control; B, a profile of EGFR levels over time was obtained by densitometric analysis and normalization for loading. Results are expressed as a percentage of EGFR levels in cortactin-overexpressing cells at 0 minutes; points, mean; bars, SE.

Receptor levels continued to decrease between 30 and 90 minutes, and by 120 minutes, the EGFR was almost undetectable. However, in cortactin-overexpressing cells, the EGF-induced down-regulation of the receptor was both delayed and impaired. Minimal down- regulation occurred in the first 60 minutes of the time course, and although receptor levels decreased between 60 and 90 minutes, significant EGFR expression persisted at 120 minutes. Cortactin increases recycling of ligand-activated epidermal growth factor receptors. Cortactin overexpression may perturb EGFR down-regulation by decreasing the internalization rate of ligand-occupied receptors, or by impairing post-internalization sorting to the lysosome, resulting in increased recycling back to the cell surface. Measurement of EGFR internalization rates did not reveal a significant difference between control cells and those overexpressing cortactin (Fig. 2A). However, following an initial phase of EGFR internalization, the reappearance of EGFRs at the cell surface was observed between 60 and 120 minutes in cortactin- overexpressing cells, presumably due to the recycling of endocy- tosed receptors (Fig. 2B). Therefore, in cortactin-overexpressing cells, a pool of internalized receptors persists which is ultimately recycled rather than degraded, an effect likely to contribute to the impaired receptor degradation at 120 minutes evident in Fig. 1B. Overexpression of cortactin does not alter ligand-induced tyrosine phosphorylation of the epidermal growth factor receptor. In order to investigate how cortactin overexpression Figure 2. The effect of cortactin overexpression on EGFR internalization and affects EGFR signaling, we first analyzed receptor autophosphor- recycling. HeLa cells were transiently transfected with a cortactin expression ylation, which is required for the recruitment of signaling proteins vector or with vector alone. Following transfection, cells were subjected to including those regulating receptor down-regulation (12, 13). Cell internalization (A) or recycling (B) assays as described in Materials and Methods. A, the internalization rates are plotted to show the ratio of internal to lysates were subjected to immunoprecipitation with an anti-EGFR cell surface receptors over time. B, the data are presented as (cpm bound at T x / antibody and the degree of receptor tyrosine phosphorylation cpm bound at T 0) Â 100; points, mean; bars, SD. www.aacrjournals.org 3275 Cancer Res 2005; 65: (8). April 15, 2005

phosphorylation and the association of c-Cbl with the EGFR was reduced in cortactin-overexpressing cells, we investigated the effect of these alterations on EGF-induced EGFR ubiquitylation. A plasmid encoding HA-tagged ubiquitin was transfected into HeLa cells in combination with either vector alone or a cortactin-encoding plasmid. Following EGF simulation, the EGFR was immunoprecipitated and analyzed for the extent of ubiquitylation by immunoblotting for the HA tag. Receptor ubiquitylation was undetectable in unstimulated cells (Fig. 4). Upon EGF treatment, EGFR ubiquitylation in control cells could be readily detected and peaked at 5 minutes of stimulation. However, overexpression of cortactin caused a substantial decrease in the level of EGFR ubiquitylation, such that this modification was undetectable at the 15-minute time point. Since sustained c-Cbl mediated ubiquitylation of the EGFR is necessary for efficient endosomal sorting and lysosomal degradation (5, 15), this provides an explanation for the impaired EGFR down-regulation in cortactin overexpressing cells (Fig. 1). Impaired epidermal growth factor receptor down-regulation upon overexpression of cortactin correlates with sustained Erk signaling. The Ras/Erk cascade is a key intracellular signaling pathway linking activation of growth factor receptors to cytoplasmic and nuclear effectors. Therefore, we investigated whether the inhibition of EGFR down-regulation by cortactin overexpression altered this downstream signaling pathway. Erk activation over a 30-minute time course was assayed by Western blotting with a phosphospecific antibody against active Erk1/2 (Fig. 5A and B). In cortactin-overexpressing and control cells, a

Figure 3. Effects of cortactin overexpression on EGFR tyrosine phosphorylation and coupling of the EGFR to c-Cbl. A, HeLa cells were transfected with a cortactin expression vector (C) or with vector alone (V) and stimulated with EGF for the indicated time period. Following lysate preparation, the EGFR was immunoprecipitated and subjected to Western blot analysis using the anti-phosphotyrosine antibody RC20 (top). The amounts of EGFR immunoprecipitated and the levels of cortactin expression in the same lysates were determined by Western blotting (middle and bottom, respectively); B, EGFR immunoprecipitates were subjected to Western blot analysis using an anti-Cbl antibody (top). Amounts of EGFR immunoprecipitated and levels of cortactin expression in the same lysates were assayed by Western blotting (middle and bottom, respectively); C, c-Cbl was immunoprecipitated and subjected to Western blot analysis with the anti-phosphotyrosine antibody RC20 (top). The amounts of c-Cbl immunoprecipitated were also determined (middle) together with levels of cortactin expression in the same lysates (bottom). A-C, representative of triplicate experiments. revealed that cortactin overexpression markedly reduced EGF- induced association of c-Cbl with the EGFR (Fig. 3B). Furthermore, Western blotting of c-Cbl immunoprecipitates with an anti- phosphotyrosine antibody revealed that EGF-induced c-Cbl tyrosine Figure 4. Cortactin overexpression attenuates EGFR ubiquitylation. HeLa cells phosphorylation was also attenuated in cortactin-overexpressing were cotransfected with a HA-tagged ubiquitin-expressing plasmid and either a cortactin expression vector (C) or with the empty vector (V). Cells were then cells (Fig. 3C), an effect which persisted until 120 minutes (data not stimulated with EGF for the indicated time periods. Following lysate preparation, shown). the EGFR was immunoprecipitated and subjected to Western blot analysis using an anti-HA antibody to detect ubiquitin-conjugated EGFR [HA (Ubiquitin), top]. An important function of c-Cbl in receptor down-regulation is to The amounts of EGFR immunoprecipitated were also determined (EGFR), ubiquitylate the EGFR (40). Tyrosine phosphorylation is required for together with levels of cortactin expression in the same cell lysates (Cortactin). the ubiquitin ligase activity of c-Cbl (13), resulting in the continuous Equivalent expression of HA-ubiquitin following the different transfections was confirmed by determining modification of proteins in total cell lysates by the ubiquitylation of the receptor and its subsequent targeting to tagged ubiquitin [HA (Ubiquitin), bottom; a representative region of the blot is lysosomal degradation (5, 6, 14, 15). Since both c-Cbl tyrosine shown]. The data are representative of triplicate experiments.

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Erk activation did not reach statistical significance until 30 minutes post-stimulation. To examine this further, HeLa cells were subjected to a more prolonged time course of EGF stimulation (Fig. 5C and D). This confirmed that cortactin overexpression leads to sustained Erk signaling, since significantly enhanced Erk activation was detected in cortactin-overexpressing cells between 30 and 120 minutes after EGF stimulation. Together, these data show that increased expression of cortactin inhibits the coupling of c-Cbl to the EGFR and thereby EGFR ubiquitylation, leading to a decrease in ligand-induced down- regulation of the receptor and sustained EGF-induced Erk activation. Similar results were obtained in COS cells (data not shown).

Figure 5. Cortactin overexpression results in sustained Erk activation in response to EGF stimulation. A, HeLa cells were transiently transfected with a cortactin expression vector (C) or with vector alone (V) and stimulated with EGF as previously described. Cell lysates were Western blotted for the activated forms of Erks 1 and 2 using a phosphorylation state-specific antibody (top). Levels of Erk and cortactin in the same lysates were determined using anti-Erk and anti-cortactin antibodies (middle and bottom, respectively); B, phospho-Erk1/2 signals were quantified by densitometry, normalized for protein loading, and represented as a percentage of maximal Erk activity (with Erk activation in cortactin-overexpressing cells at 2 minutes of stimulation treated as 100%). Points, mean; bars, SE; C and D, analysis of Erk activation over a prolonged time course was analyzed as for (A) and (B); D, Erk activation in cortactin-overexpressing cells at 30 minutes of stimulation is treated as 100%. Columns, mean; bars, SE; B and D, *, P < 0.02; **, P V 0.005 by unpaired Figure 6. Determination of the rates of EGFR down-regulation in a panel of Student’s t test, indicating significant differences between HNSCC cell lines. A, EGFR and cortactin expression in HNSCC cell lines. cortactin-overexpressing and control cells. Equivalent amounts of cell lysate were Western blotted as indicated, with h-actin providing a loading control; B, EGF-induced down-regulation of the EGFR and Erk activation in a high (FaDu) and low (SCC25) cortactin-expressing cell line. Cells were serum-starved and then stimulated with EGF for the indicated times. similar increase in Erk activation relative to unstimulated cells was Cell lysates were prepared and Western blotted as indicated; C, quantitative evident after 2 minutes of stimulation. However, in cortactin- analysis of EGFR down-regulation in HNSCC cell lines. Analyses were done as overexpressing cells, Erk activity then decayed more slowly. in (B), and a profile of EGFR levels over time was obtained by densitometric analysis and normalization for loading. Results are expressed as a percentage of Reflecting this, there was a trend for Erk phosphorylation in these EGFR levels at 0 minutes; points, mean; bars, SE. To simplify the graph, only cells to be higher at 5 and 15 minutes, although the difference in the upper half of error bars are shown, i.e., above the data points. www.aacrjournals.org 3277 Cancer Res 2005; 65: (8). April 15, 2005

562, which are 11q13-amplified, we categorized FaDu, Detroit 562, and SCC15 as cortactin-overexpressing HNSCC cell lines. SCC9 and SCC25 served as low-expressing controls. Interestingly, analysis of ligand-induced EGFR down-regulation rates revealed that the cell lines segregated into the same two groups as previously defined by their cortactin expression status. In SCC9 and SCC25 cells, which express low amounts of cortactin, receptor down-regulation was rapid, with only 10% to 20% of the receptor remaining after 90 minutes (Fig. 6B and C). In contrast, receptor down-regulation was much slower in the cortactin- overexpressing FaDu, Detroit 562, and SCC15 cell lines. In these lines, 60% to 80% of the EGFR persisted at the end of the time course. Furthermore, Erk activation was also more sustained in cortactin-overexpressing HNSCC cells (Fig. 6B and data not shown). These data cannot be explained by differences in EGFR expression, since EGFR levels are comparable in SCC9 cells, which exhibit rapid receptor degradation, and FaDu cells, in which EGFR down-regulation is attenuated (Fig. 6A). Although there was an inverse correlation between the levels of cortactin expression in this panel of HNSCC cell lines and the rates of ligand-induced EGFR down-regulation, it was possible that other differences between the cell lines could underlie this observation. Although heterodimerization of the EGFR with erbB2 results in enhanced recycling of the former receptor (18), this does not explain the differences between the HNSCC cell lines, as SCC9 cells express more erbB2 than Detroit 562s, and comparable levels to the FaDu line (data not shown). Rather than investigating further alternative explanations for the differential rates of EGFR degradation, we decided to directly test the role of cortactin. First, we overexpressed cortactin in SCC9 cells. This reduced EGF- induced receptor degradation at 60 and 90 minutes of stimulation, and led to more sustained Erk activation (Fig. 7A, representative of triplicate experiments). Second, in order to determine the contribution from cortactin in 11q13-amplified cells, we sup-

Figure 7. Cortactin overexpression contributes to attenuation of EGFR pressed cortactin expression in Detroit 562 cells by transfection of down-regulation in HNSCC cells. A, SCC9 cells were transiently transfected with a selective siRNA (27). Although we were unable to reduce a cortactin expression vector or with vector alone, and then stimulated with EGF cortactin levels to those of the low-expressing lines, we achieved for the indicated time period. Cell lysates were prepared and Western blotted as indicated. The data is representative of triplicate experiments; B, Detroit 562 a level of expression midway between SCC25 and SCC15 cells cells were transfected with a cortactin-selective siRNA or a lamin A/C-selective (Fig. 7B). Strikingly, this accelerated ligand-induced EGFR down- siRNA as a control, and then stimulated with EGF for the indicated time periods. regulation and attenuated Erk activation, as indicated by a Cell lysates were prepared and Western blotted as indicated. The data is representative of triplicate experiments. significant reduction in Erk phosphorylation at the 90-minute time point (Fig. 7B, representative of triplicate experiments). Similar results were obtained with different cortactin-selective siRNAs Cortactin overexpression attenuates ligand-induced epi- (ref. 37 and data not shown). Therefore, although other factors dermal growth factor receptor down-regulation in head and probably also contribute to the contrasting receptor down- neck squamous cell carcinoma cell lines. In the previous regulation profiles among HNSCC cell lines, these data clearly experiments, we utilized HeLa cells as a model system. In order show that cortactin overexpression in HNSCC cells acts to to investigate whether comparable effects of cortactin could be attenuate EGFR down-regulation and prolong Erk activation, and detected in cancer cells exhibiting EMS1 amplification and/or identify a novel functional role for cortactin in human cancers. overexpression of endogenous cortactin, we utilized a panel of HNSCC cell lines. This cell type was chosen since HNSCCs commonly exhibit amplification of the 11q13 locus in which the Discussion EMS1 gene resides (30). The panel comprised five cell lines; Aberrant signaling by receptor tyrosine kinases has been FaDu and Detroit 562, which feature 11q13 amplification, and associated with many human cancers, and escape from c-Cbl- SCC9, SCC15 and SCC25, in which the 11q13 copy number is mediated receptor down-regulation has recently been shown to relatively normal (34, 41). Determination of cortactin expression be an important event in receptor deregulation (1). In this study, in this panel by Western blot analysis revealed that FaDu cells we show that cortactin overexpression impairs c-Cbl phosphor- express the highest levels of cortactin, approximately 6-fold ylation and association with the EGFR and attenuates ligand- higher than the lowest expressing line, SCC9 (Fig. 6A). The next induced EGFR down-regulation. This results in sustained EGFR highest expressors were Detroit 562 cells and SCC15 cells. Since signaling in cortactin-overexpressing cells and provides a novel SCC15 cells expressed comparable levels of cortactin to Detroit explanation for EMS1 gene amplification in human cancers.

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Tyrosine phosphorylation of c-Cbl stimulates its ubiquitin ligase either degraded, or recycled back to the surface. The degradation activity and CIN85 binding, which are both implicated in ligand- observed could be triggered by low-level ubiquitylation of the induced receptor down-regulation (4, 7, 8). In this study, inhibition receptor by c-Cbl or another ubiquitin ligase, and/or other sorting of c-Cbl tyrosine phosphorylation by cortactin overexpression had mechanisms. Interestingly, a similar delay in ligand-induced EGFR no significant effect on receptor internalization. Presumably, either degradation was recently observed in c-Cbl-deficient MEFs (5). It is sufficient CIN85 is recruited, or other endocytic pathways likely that a persistence of the EGFR in particular endosomal compensate, to allow normal ligand-induced receptor internaliza- compartments underlies the sustained Erk activation in cortactin- tion to occur when cortactin is overexpressed. However, receptor overexpressing cells, since endosomal EGFRs play an important ubiquitylation was markedly impaired, resulting in an increase in role in stimulation of the Shc/Ras/Erk pathway (49, 50). Since we receptor recycling at late time points. Consistent with these did not detect significant EGFR degradation in HeLa cells until 30 observations, analyses of c-Cbl knockout fibroblasts and Chinese minutes of EGF stimulation (Fig. 1 and data not shown), the hamster ovary cells conditionally defective in ubiquitylation show a modest enhancement of Erk activation at 5 and 15 minutes of EGF requirement for c-Cbl-mediated receptor ubiquitylation in late stimulation in cortactin-overexpressing cells (Fig. 5) may reflect endosomal sorting and lysosomal degradation of the EGFR, but not altered trafficking of the EGFRs among specific endosomal receptor internalization (5). Also, expression of ubiquitylation- compartments. This is currently under investigation. defective mutants of Cbl results in impaired ubiquitylation of Interestingly, the EGFR is implicated in the progression of head receptor tyrosine kinases, reduced lysosomal targeting and and neck cancers and estrogen receptor–negative breast cancers enhanced receptor recycling and signaling (4, 13, 40). Therefore, (51), both cancers where EMS1 amplification has prognostic impaired receptor ubiquitylation due to reduced c-Cbl activation significance (29, 30). This highlights the possibility that amplifica- and coupling to the EGFR represents the most likely explanation tion of EMS1 in primary tumors may provide a proliferative for the observed effect of cortactin overexpression on EGFR down- advantage via enhanced EGFR mitogenic signaling, with effects on regulation and recycling. cellular invasion and metastasis functioning later in tumor The mechanism for the observed effects of cortactin over- progression (52). Studies using fibroblasts, hepatocellular carcino- expression on c-Cbl function are unclear. The activity of c-Cbl ma or MDA-MB-231 breast cancer cells have not detected effects of towards the EGFR is regulated by both protein-protein interactions cortactin overexpression on proliferative end-points (31, 33, 53). and tyrosine phosphorylation. For example, Sprouty2 attenuates However, these studies did not specifically examine EGF-induced EGFR ubiquitylation and down-regulation by binding to the SH2 responses, and in the case of MDA-MB-231 breast cancer cells, the domain of c-Cbl and inhibiting its association with the EGFR (42). presence of mutant K-Ras and B-Raf proteins may mask any effect However, we did not observe an increase in Sprouty2 expression or of cortactin on Erk activation (54). It is also possible that cortactin association with c-Cbl in cortactin-overexpressing cells (results not overexpression requires additional events during tumor progres- shown). Also, activated Cdc42 binds to c-Cbl via p85Cool-1 (for sion to exert mitogenic effects. Finally, it should be noted that cloned-out-of-library)/h-Pix (for Pak-interactive exchange factor) potentiation of Erk signaling in cortactin-overexpressing cells may and inhibits the coupling of c-Cbl to the EGFR and thus receptor also contribute to the known role of cortactin in cell migration, ubiquitylation (43). Fgd1, a Cdc42 guanine nucleotide exchange since phosphorylation of cortactin by Erk has recently been shown factor, directly interacts with and is targeted within the cell by to increase the ability of cortactin to enhance N-WASP-mediated cortactin (44). Therefore, overexpression of cortactin may activate actin polymerization (55). Increased Erk activation may also Cdc42 via Fgd1, leading to Cdc42-h-Pix-c-Cbl complex formation promote cell migration through effects on focal adhesion dynamics and reduction of the interaction of c-Cbl with the EGFR. Finally, the and activation of myosin light chain kinase (56). Therefore, nonreceptor tyrosine kinase Src phosphorylates and activates the enhanced EGF-induced Erk signaling due to cortactin over- ubiquitin ligase activity of c-Cbl, leading to ubiquitylation of Src expression could contribute to the multifaceted process of tumor itself and c-Cbl (45, 46). Inhibition of Src kinases using the specific progression by affecting both the proliferation and motility of inhibitor PP1 impairs c-Cbl phosphorylation and association with cancer cells. the EGFR leading to reduced ubiquitylation of the receptor (47). Since cortactin is a known Src substrate and also binds to this Acknowledgments tyrosine kinase (48), it is possible that overexpression of cortactin in cells may act to sequester Src from c-Cbl and thereby inhibit c- Received 6/15/2004; revised 1/23/2005; accepted 2/1/2005. Grant support: Department of Defense Breast Cancer Research Program grant Cbl phosphorylation and function towards the EGFR. The roles of DAMD17-00-1-0251, which is managed by the U.S. Army Medical Research and Cdc42 and Src in the observed effects of cortactin are currently Materiel Command, the National Health and Medical Research Council of Australia, and the Cancer Council New South Wales. under investigation. The costs of publication of this article were defrayed in part by the payment of page Based upon the receptor down-regulation, internalization, and charges. This article must therefore be hereby marked advertisement in accordance recycling data for cortactin-overexpressing cells presented in Figs. 1 with 18 U.S.C. Section 1734 solely to indicate this fact. We thank Stephanie Winata for technical assistance, Dr. Larry Kalish for advice and 2, internalized EGFRs accumulate until an apparent threshold regarding the HNSCC cell lines, and Prof. David Drubin (University of California) and is reached at 60 minutes. At this point, the receptors begin to be his laboratory for assistance with design of cortactin-selective siRNAs.

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