Cortactin Overexpression Regulates Actin-Related Protein 2/3 Complex Activity, Motility, and Invasion in Carcinomas Withchromosome11q13 Amplification Brian L

ResearchArticle

Cortactin Overexpression Regulates Actin-Related Protein 2/3 Complex Activity, Motility, and Invasion in Carcinomas withChromosome11q13 Amplification Brian L. Rothschild,1,2 Ann H. Shim,1 Amanda Gatesman Ammer,4 Laura C. Kelley,4 Kimberly B. Irby,1 Julie A. Head,1 Lin Chen,3 Marileila Varella-Garcia,3 Peter G. Sacks,5 Barbara Frederick,2 David Raben,2 and Scott A. Weed1,4

Departments of 1Craniofacial Biology, 2Radiation Oncology, and 3Medical Oncology, University of Colorado at Denver and Health Sciences Center, Aurora, Colorado; 4Mary Babb Randolph Cancer Center, Department of Neuroscience and Anatomy, West Virginia University, Morgantown, West Virginia; and 5Department of Basic Science and Craniofacial Biology, College of Dentistry, New York University, New York, New York

Abstract edge of the cell, resulting in the production of polarized Carcinoma cell motility and invasion are prerequisites lamellipodia that are oriented toward the direction of movement. for tumor cell metastasis, which requires regulation of the Production and extension of lamellipodia occurs in response to a actin cytoskeleton. Cortactin is an actin-related protein 2/3 wide variety of extracellular ligands that signal through trans- (Arp2/3)complex–activating and filamentous (F)-actin–bind- membrane receptors (1). Lamellipodia serve to orient tumor cells ing protein that is implicated in tumor cell motility and toward the vasculature in response to chemotatic agents and are metastasis, partially by its ability to become tyrosine required for tumor cell invasion (2). phosphorylated. Cortactin is encoded by the CTTN gene and Chemotatic receptor activation results in increased Rho GTPase maps to chromosome 11q13, a region amplified in many and Src family kinase activity, which in turn leads to the activation carcinomas, including head and neck squamous cell carcino- of Wiskott-Aldrich syndrome proteins (WASp) that drive lamelli- ma (HNSCC). CTTN gene amplification is associated with podia protrusion (3). Activation of WASps by GTPase activation or lymph node metastasis and poor patient outcome, and tyrosine phosphorylation exposes a cryptic carboxyl-terminal cortactin overexpression enhances motility in tumor cells domain containing an acidic region that binds and activates the lacking 11q13 amplification. However, a direct link between actin-related protein 2/3 (Arp2/3) complex. Arp2/3 complex increased motility and invasion has not been reported in activity results in nascent polymerization of actin filaments at tumor cells with chromosome 11q13 amplification and the cell periphery, providing the protrusive force responsible for cortactin overexpression. In this study, we have examined lamellipodia extension at the leading edge (4). Regulation of the the relationship between CTTN amplification and tumor cell actin cytoskeleton by Arp2/3 complex activity has been proposed motility in HNSCC. In 11 of 39 (28%)HNSCC cases, cortactin as a mechanism for controlling tumor cell migration, invasion, and overexpression determined by immunohistochemistry corre- metastasis (5). lates with lymph node metastasis and CTTN gene amplifica- In addition to WASps, another protein that binds and activates tion. HNSCC cells containing cortactin gene amplification and Arp2/3 complex is cortactin, a Src kinase substrate and filamentous protein overexpression display increased binding and activa- (F)-actin–binding protein that is enriched in lamellipodia (6, 7). tion of Arp2/3 complex, and were more motile and invasive Cortactin consists of an amino-terminal acidic domain followed by than HNSCC cells lacking CTTN amplification. Down-regula- a repeat region, a proline-rich region containing sites of tyrosine tion of cortactin expression in CTTN-amplified HNSCC cells by and serine phosphorylation, and a distal Src homology 3 (SH3) small interfering RNA impairs HNSCC motility and invasion. domain that interacts with a variety of proline-rich proteins (6, 7). Treatment of HNSCC cells with the epidermal growth factor Cortactin directly activates Arp2/3 complex actin nucleation receptor inhibitor gefitinib inhibits HNSCC motility and activity through the binding of the amino-terminal acidic domain down-regulates cortactin tyrosine phosphorylation. These region to Arp2/3 complex and the repeat region to F-actin (7). data suggest that cortactin may be a valid prognostic and Nucleation activity and lamellipodia protrusion is further enhanced therapeutic marker for invasive and metastatic HNSCC and by the binding of the SH3 domain to dynamin 2 or WASp- other carcinomas with 11q13 amplification. (Cancer Res 2006; interacting protein (8, 9). The cortactin SH3 domain also binds 66(16): 8017-25) and activates neuronal (N)-WASp, which in turn promotes Arp2/3 actin nucleation and cell motility (10, 11). Additionally, cortactin Introduction modulates cell movement by the phosphorylation of three specific tyrosine residues (421, 470, and 486 in humans) by Src and other Tumor cell motility is required for local-regional invasion and oncogenic tyrosine kinases (12). Tyrosine-phosphorylated cortactin distant metastasis. Motility is initiated by protrusion of the leading is found in lamellipodia (13) and correlates with enhanced cell motility and tumor cell metastasis in nude mice (14). Other functions of cortactin include stabilization of Arp2/3–F-actin Note: B.L. Rothschild and A.H. Shim contributed equally to this work. Requests for reprints: Scott A. Weed, Mary Babb Randolph Cancer Center, West networks and participation in receptor-mediated endocytosis Virginia University, Morgantown, WV 26508-9300. Phone: 304-293-3016; Fax: 304-293- (15, 16). 4667; E-mail: [email protected]. I2006 American Association for Cancer Research. Genomic instability is a hallmark of cancer progression and doi:10.1158/0008-5472.CAN-05-4490 results in the accumulation of specific genetic events. Amplification www.aacrjournals.org 8017 Cancer Res 2006; 66: (16). August 15, 2006

Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2006 American Association for Cancer Research. Cancer Research of chromosome 11q13 is found in several tumors and is one of the amplification in HNSCC cell lines, mitotic arrest was achieved by treating most prevalent genetic alterations in head and neck squamous cell the cells with colcemid (0.05 Ag/mL) for 2 hours and chromosome A carcinoma (HNSCC), occurring in approximately one third of all elongation by treatment with ethidium bromide (5 g/mL) for 1 hour. Cells cases (17). Amplification of 11q13 correlates with several important were trypsinized, lysed with 75 mmol/L potassium chloride, and fixed with 3:1 (v/v) mixture of methanol and acetic acid. Cells were dropped on glass clinical variables, including increased invasive disease, regional slides and probes were hybridized and processed as described (28). The metastasis, more frequent recurrence, and increased disease- number of CTTN and CEP 11 probe signals were determined for a minimum CTTN specific mortality (18). The human cortactin locus (formerly of 100 nuclei in each cell line by epifluorescence microscopy. EMS1 designated ) is located within the 11q13 region and cortactin Cell lines, transfection, and Western blotting. HNSCC lines UMSCC2, overexpression resultant from gene amplification is found in UMSCC8, UMSCC10A, UMSCC19, and HN4 were maintained in DMEM HNSCC and other carcinomas (19, 20). CTTN amplification and (Mediatech, Herndon, VA) containing 10% fetal bovine serum (FBS; overexpression directly correlates with poor outcome in HNSCC HyClone, Logan, UT), 50 Ag/mL penicillin-streptomycin, and nonessential patients (21). Based on these data, it has been proposed that cor- amino acids. The MSK921 and 584 lines were cultured in L-glutamine–free tactin is the critical gene in the amplified chromosome 11q13 region RPMI (Mediatech) supplemented with 10% FBS and 50 Ag/mL penicillin- 6 A responsible for enhancing carcinoma motility and invasion (22). streptomycin. For transfection, 10 cells were incubated with 2 g FITC- Indirectly supporting this, several recombinant overexpression labeled control small interfering RNA(siRNA);siRNAtargeted against human cortactin (Qiagen, Valencia, CA); pEGFPN1 green fluorescent protein studies in cells lacking 11q13 amplification have established (GFP) vector (BD Clontech, Palo Alto, CA); GFP-cortactin (9); FLAG-wild cortactin as key regulator in migration, invasion, and metastasis type or FLAG-Y421F, FLAG-Y466F, FLAG-Y482F [triple point mutant (TPM)] (12, 23, 24). To determine the role cortactin plays in motility and cortactin (13) in solution V (Amaxa Biosystems, Berlin, Germany); and invasion in the context of tumors with 11q13 amplification, we transfected with the Nucleofector I device (Amaxa Biosystems) according to have investigated the effect of cortactin overexpression in HNSCC the instructions of the manufacturer. Cells were plated and allowed to cells with and without 11q13 amplification and have determined recover for 24 to 48 hours before use in motility, invasion, or Western blot that cortactin overexpression derived from 11q13 amplification experiments. Cell lysis and Western blot analysis was done as previously results in increased Arp2/3 complex activity, tumor cell motility, described (13). The following antibodies and final concentrations were used: A and invasion. We also show that cortactin tyrosine phosphorylation 4F11 (0.5 g/mL), anti-ARPC2 (p34 subunit of Arp2/3 complex, 1:1,000; h is required for efficient HNSCC migration, and that inhibition of Upstate, Waltham, MA), anti- -actin (1:5,000; Calbiochem, La Jolla, CA), Living Colors anti-GFP clone JL-8 (1:1,000; BD Clontech), and antiphospho- cortactin tyrosine phosphorylation with the targeted therapeutic tyrosine 4G10 (1:1,000; Upstate). Scanned blots were quantified using drug gefitinib reduces HNSCC motility and corresponds with ImageQuant TL software (Amersham Biosciences, Piscataway, NJ). Blots decreased cortactin tyrosine phosphorylation. These results indi- were done in triplicate. cate that cortactin directly contributes to elevated motility and Arp2/3 pulldown and actin incorporation assays. The recombinant invasion in HNSCC tumors with 11q13 amplification by increasing glutathione-S-transferase (GST)-verprolin cofilin homology acidic (VCA) Arp2/3 actin nucleation activity, and that cortactin tyrosine domain of N-WASp was expressed and purified using standard techniques. phosphorylation may serve as a potential diagnostic marker for Cell lysates (1 mg) were incubated with 50 Ag GST-VCAprebound to 40 AL validation of targeted therapeutic agents against tyrosine kinase glutathione-agarose (50% slurry; Pierce, Rockford, IL) for 3 hours at 4jC, pathways involved in tumor cell migration and invasion. washed, and processed for Western blotting with 4F11 and anti-ARPC2 antibodies. Assays were done in triplicate. Globular (G)-actin incorporation (barbed end) assays on serum-starved cells were done essentially as described (29), except Alexa-488–G-actin (Invitrogen, Carlsbad, CA) was Materials and Methods used and cells were stimulated for 10 minutes with 100 ng/mL epidermal Tumor samples. Formalin-fixed, paraffin-embedded tissue blocks from growth factor (EGF; Upstate). Fixed cells were imaged with a Nikon TE2000 10 cases of HNSCC were retrieved from the University of Colorado Health epifluoresence microscope equipped with a Cool-Snap f/x charge-coupled Sciences Center Department of Pathology archives. All tissue and case device camera (Roper Scientific, Tucson, AZ). Quantification of actin report information was used under approval from the Colorado Multiple incorporation was done using a script written in the Metamoph v6.0 Institutional Review Board. An additional 29 HNSCC cases were analyzed software suite (Molecular Devices, Downingtown, PA). The script identifies from a HNSCC tissue microarray (YTMA7) constructed at Yale University peripheral areas around the cell with pixel intensities above background Department of Pathology. and creates a 3-pixel-thick ring over the regions of circumferential Immunohistochemistry. Five-micrometer sections cut from tissue fluorescent actin incorporation. Border widths were adjusted manually as blocks and the HNSCC microarray were processed for immunohistochem- required to compensate for varying degrees of cortical actin thickness. ical labeling as described (25), except that sections were incubated with Quantification of G-actin incorporation was taken as the total fluorescence 0.4% pepsin for 10 minutes at 37jC for antigen retrieval. The pan-specific intensity divided by the surface area of the measured region. Fifty EGF- cortactin monoclonal antibody 4F11 and avian-specific control cortactin stimulated and nonstimulated cells for each line were analyzed in three antibody 1H3 (26) were used at 1 Ag/mL. Samples were visualized with an independent experiments. Olympus AX70 microscope and images were acquired using the Micro- Cell motility and invasion assays. Transwell cell migration and BrightField system (Williston, VT). Matrigel-coated invasion assays were conducted using 8 Am pore size Fluorescence in situ hybridization. Dual-color fluorescence in situ chambers (Fisher Scientific, Pittsburgh, PAand BD Transduction, San hybridization (FISH) analysis was conducted to determine CTTN copy Jose, CA) essentially as described (30). Cells (105) were added to each upper numbers in tumor specimens and in HNSCC cell lines. Two P1 genomic chamber and cells were allowed to migrate or invade for 24 hours. In some cortactin clones (20) were used in combination with a control probe for the experiments, 1 Amol/L gefitinib (Iressa, AstraZeneca, Macclesfield, Chesh- centromeric region of chromosome 11 (CEP 11 SpectrumGreen, Vysis/ ire, United Kingdom) was added to both chambers at the beginning of Abbott Molecular, Des Plaines, IL). The CTTN P1 clones were labeled by the experiment. Fixed cells were stained with 3 Ag/mL 4¶,6-diamidino-2- nick translation with dUTP-conjugated SpectrumRed, combined with the phenylindole (DAPI) and motility was assessed by counting nuclei in four SpectrumGreen CEP 11, and hybridized as described (27). random microscopic fields through the use of an automated Metamorph Tumor specimens were quantitated for CTTN gene amplification by script that accurately determines cell count by delineating cell shapes and epifluorescence, with cases containing a ratio of >2 CTTN/CEP 11 copies dividing by the average nuclear surface area. For siRNArescue experiments, per nucleus considered amplified. For determining cortactin gene cell motility was measured using electric substrate impedance sensing

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(Applied BioPhysics, Troy, NY), as described (31), using 40 kHz voltage pulse sections treated with a monoclonal antibody specific to avian for initial cell ablation and 10 ng/mL EGF as the motile stimulus. cortactin as a control were not significantly stained (Fig. 1A). All Impedance was measured for 24 hours. Aminimum of three independent tumors that overexpressed cortactin also contained CTTN experiments were done in all assays. amplification as determined by FISH (Fig. 1B). Noninvasive, Statistical analysis. P values were determined by the unpaired, two- node-negative tumors had little cortactin staining and did not tailed, Student’s t test. contain CTTN amplification (Fig. 1). These data indicate that cortactin overexpression and gene amplification in HNSCC Results correlates with an invasive phenotype. Cortactin overexpression in invasive HNSCC. Although Cortactin overexpression and gene amplification correlate cortactin gene amplification and transcription has been docu- in HNSCC cells. To determine the relationship between cortactin mented with PCR-based methods in HNSCC and correlates with overexpression and gene amplification in HNSCC cells, we poor prognosis (21), cortactin localization and CTTN amplification quantified cortactin protein and gene levels in several HNSCC cell status in HNSCC have not been visualized and correlated to lines where cortactin expression and/or chromosome 11q13 am- invasive capacity. To address this, we analyzed cortactin expression plification status had been partially defined (Fig. 2; refs. 32–34). in 39 cases of primary HNSCC by immunohistochemistry and Cortactin protein and gene levels were standardized against CTTN amplification by FISH analysis from archived tissue sections UMSCC10A, a line normal for cortactin expression compared with and a HNSCC tissue microarray (Fig. 1). In 11 cases (28%), cortactin nontransformed epithelial cells (not shown) and disomic for CTTN. overexpression was detected in the tumor cell cytoplasm, with Western blot analysis indicated a 6- to 12-fold increase in cortactin marked enrichment of the protein at the cell periphery and at the expression for lines UMSCC19, UMSCC2, and MSK921 (Fig. 2A leading edge of cells in tumors that displayed local-regional and B). The HN4 line had a 2-fold increase in cortactin expression, invasion and cervical lymph node metastasis (Fig. 1A). Serial whereas UMSCC8 and UMSCC584 contained normal cortactin levels (Fig. 2A and B). The amount of Arp2/3 complex for each line was also determined with an antibody against the ARPC2 complex subunit. In all lines, no significant differences in Arp2/3 expression were detected (Fig. 2A). Cytogenetic analysis in metaphase spreads indicated that these lines were hypo-diploid to near-triploid for CTTN. Quantification of the CTTN locus by FISH indicated that UMSCC19, UMSCC2, and MSK921 contained clustered CTTN amplification, with mean copy numbers ranging from 8 to over 20 per cell (Fig. 2C). All other lines had on average two to four CTTN copies per cell. With the exception of the HN4 line, the level of cortactin protein expression directly correlates with CTTN copy number in each line (Fig. 2B and C). These data indicate that cortactin expression and gene levels are tightly coupled, and that amplification of CTTN correlates with cortactin overexpression in HNSCC. HNSCC cells containing cortactin overexpression have increased Arp2/3 binding and activation. Our findings that cortactin is enriched at the cell periphery when overexpressed in invasive tumors and that Arp2/3 levels are unchanged suggests that cortactin overexpression resulting from chromosome 11q13 amplification may increase the amount of cortactin associated with Arp2/3 complex, resulting in enhanced Arp2/3 actin nucleation activity. To test this possibility, we conducted Arp2/3 pulldown experiments from HNSCC cells with and without cortactin overexpression using a N-WASp recombinant GST-VCA domain, a region of N-WASP that simultaneously binds with cortactin to different subunits of Arp2/3 (35). A 1.5- to 2-fold increase in cortactin binding to Arp2/3 complex was observed in cell lines Figure 1. Immunohistochemical and FISH analysis of cortactin overexpression and CTTN amplification in invasive HNSCC. A, serial sections of tumor samples UMSCC2 and UMSCC19 that overexpress cortactin compared with from two patients (invasive-1 and invasive-2) with node-positive, invasive UMSCC10Aand UMSCC8 (Fig. 3 A). Asimilar increase in binding HNSCC, and sections from one patient (non-invasive) with node-negative, was observed in the overexpressing line MSK921 (data not shown). noninvasive HNSCC were processed for immunohistochemistry with pan-species reactive (cort IHC) or chicken-specific (ctl) cortactin monoclonal These results suggested that the increased association between antibodies and counterstained with hematoxylin. Arrowheads, areas of cortical Arp2/3 and cortactin may result in increased cortactin-mediated cortactin enrichment. Bar, 100 Am. B, analysis of cortactin overexpression Arp2/3 actin nucleation activity. To test this, serum-starved HNSCC and CTTN copy levels in a HNSCC tissue microarray. Top, cortactin overexpression was determined by immunohistochemistry as in (A, cort IHC), cells were permeabilized with saponin in the presence of Alexa-488– with overexpression shown for two representative cases (amp-1 and amp-2) G-actin and then stimulated with EGF to allow the incorporation of and normal expression shown in a third case (disomic). Arrowheads, cortical B C localization. Bottom, determination of CTTN levels by FISH (cort FISH). labeled actin into nascent filament ends (Fig. 3 and ). This A serial section of the HNSCC microarray was hybridized with probes against method allows for determining the degree of Arp2/3 actin- CTTN (red) and the centromeric region of chromosome 11 (green). Blue, nuclei nucleation activity within newly forming lamellipodia downstream visualized by DAPI staining. Arrows, regions of clustered amplification for the cortactin gene. Bar for immunohistochemistry images, 100 Am. Bar for FISH of EGF receptor (EGFR) activation (36). In all HNSCC lines images, 20 Am. examined, maximal incorporation of Alexa-488–G-actin into the www.aacrjournals.org 8019 Cancer Res 2006; 66: (16). August 15, 2006

Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2006 American Association for Cancer Research. Cancer Research peripheral cell edge occurred 10 minutes after EGF stimulation, MSK921 and UMSCC19 lines showed a nearly 2-fold increase in resulting in circumferential rings of varying fluorescence intensities cortical actin polymerization compared with the UMSCC8 and 584 (Fig. 3B). Levels of EGFR were similar in all lines examined (data not lines (Fig. 3C). Similar results were observed in UMSCC2 cells, shown). Quantitative microscopic analysis of this region for the although precise localization of labeled actin within lamellipodia was difficult to discern in this cell line (data not shown). These data indicate that HNSCC cells with cortactin overexpression are enhanced in their ability to bind and activate Arp2/3 complex at the cell periphery. HNSCC cells with 11q13 amplification display increased cell motility and invasion. To determine if HNSCC cells with 11q13 amplification and cortactin overexpression display increased cell motility and invasion, transwell migration and Matrigel invasion assays were conducted (Fig. 4). No significant changes in cell division rates were observed between all lines during the assay, allowing for direct comparisons to be made. Two different chemo- attractants, EGF and FBS, were evaluated because all HNSCC lines used overexpress EGFR (except UMSCC10A) and EGFR overexpression is clinically known to correlate with increased HNSCC motility and invasion (37). Increased migration and invasion was observed with UMSCC19, UMSCC2, and MSK921 cells toward 10 ng/mL EGF compared with all other lines tested (Fig. 4). Similar results were obtained when 10% FBS was used as the chemoattractant, with some variability in the motility and/or invasive response observed in the UMSCC8 and MSK921 lines when compared with EGF (Fig. 4). Collectively, these data suggest that 11q13 amplification and cortactin overexpression correlates with increased HNSCC motility and invasion. Cortactin directly modulates HNSCC motility and invasion. The 11q13 locus is a gene-rich region that upon amplification may express proteins besides cortactin that can contribute to cell motility and invasion in HNSCC. To determine the contribution of cortactin in HNSCC motility and invasion, cortactin expression was down-regulated in HNSCC cells with 11q13 amplification by transfection with a cortactin-specific siRNA. The degree of cortactin knockdown varied between the different cell lines tested, ranging to near-complete knockdown for UMSCC2 (97%), 83% for UMSCC19, and 40% for MSK921 (Fig. 5A). This variation is reflective of differing transfection efficiencies among the tested lines as determined by control transfections with a plasmid encoding GFP (data not shown). UMSCC2 cells were inhibited in motility 47% toward EGF and 43% serum when compared with internal, cell line–specific controls (Fig. 5B). The corresponding values for UMSCC19 were 51% and 52%; for MSK921, the values were 34% and 9%, respectively. Invasion was also inhibited in cells with cortactin knockdown, with a 29% reduction for UMSCC2, 21% for UMSCC19, and 10% for MSK921 with EGF and the motile stimulus. These values were 43%, 65%, and 14%, respectively, when FBS was used (Fig. 5B). To further confirm that cortactin expression levels influence HNSCC motility Figure 2. Determination of cortactin protein and CTTN levels in HNSCC cell lines. A, Western blot analysis of cortactin and Arp2/3 complex expression. and invasion, UMSCC8 and HN4 cells (lines without 11q13 Equal amounts of cell extracts from HNSCC cell lines (top) were subjected to amplification) were transfected with a construct expressing either SDS-PAGE and Western blotted with antibodies against cortactin, against the GFP or GFP-cortactin. Expression of GFP-cortactin in these cells ARPC2 subunit of Arp2/3 complex, and against h-actin as a loadingcontrol. B, quantification of protein levels in HNSCC cell lines. Western blots described raised the total cortactin levels to 125% in UMSCC8 and 140% in in (A) were scanned and quantified with the cortactin/h-actin ratio for each HN4 (Fig. 5C). Motility and invasion for both lines expressing GFP- cell line determined for three independent experiments. Columns, averaged cortactin was increased 15% to 63% above the values for internal, values normalized to the UMSCC10A cortactin/h-actin values (designated as 1.0) for control purposes; bars, SE. *, P < 0.05. C, CTTN levels in HNSCC cell GFP-expressing controls (Fig. 5D). These values are similar to what lines. Fixed cells were analyzed by FISH usinga combination of two independent has been reported in other cell systems with similar methodology probes containinggenomic CTTN sequences conjugated to SpectrumRed. The chromosome 11 centromere was visualized usinga specific centromeric (23). Taken together, these data indicate that cortactin over- probe conjugated to SpectrumGreen. Columns, average number of CTTN genes expression directly results in enhanced HNSCC motility and per cell, for lines with normal or near-normal ploidy levels (UMSCC10A, invasion in tumor cells with 11q13 amplification. UMSCC8, UMSCC584, and HN4). For cell lines with clustered cortactin CTTN amplification (UMSCC2, UMSCC19, and MSK921), the lower limit of each Gefitinib inhibits HNSCC motility and down-regulates cortac- average value is shown. Bars, SE. *, P < 0.001. tin tyrosine phosphorylation. Cortactin tyrosine phosphorylation

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Figure 3. Cortactin overexpression in HNSCC enhances Arp2/3 actin nucleation activity. A, enhanced association of Arp2/3 complex in tumor cells overexpressingcortactin. Top, 1 mgtotal cell extracts from the indicated cell lines was incubated with GST-N-Wasp VCA domain prebound to glutathione agarose beads, pelleted, and washed. Bound proteins were detected by Western blottingwith antibodies against cortactin and the ARPC2 subunit of Arp2/3 complex. Bottom, quantification of cortactin associated with Arp2/3 complex. Western blots were quantified and the cortactin/ARPC2 ratio was determined for each cell line for three independent experiments. Columns, averaged values normalized to the UMSCC10A/ ARPC2 values (designated as 1.0 for control purposes); bars, SE. *, P < 0.05. B, increased EGF-mediated actin polymerization in HNSCC cells overexpressingcortactin. Cell lines containingnormal cortactin levels (UMSCC8 and UMSCC584) or cortactin overexpression (MSK921 and UMSC19) were plated onto fibronectin-coated coverslips, serum starved, and either mock-treated (ÀEGF)or stimulated with EGF (+EGF) in the presence of saponin and Alexa 488–conjugated G-actin. Cells were fixed and visualized for actin incorporation (Actin). Images were evaluated for cortical actin incorporation by the application of a 3-pixel-wide border programmed to cover peripheral areas of actin incorporation and calculate the average pixel intensity of the entire border (orange outline, Actin+Periphery). Representative images for lines UMSCC 8 and UMSCC 19. C, quantification of peripheral actin incorporation. Columns, average pixel intensity for the cortical regions, quantified as described in Materials and Methods; bars, SE, *, P < 0.02.

is required for efficient motility and metastasis (12, 14). EGFR and phorylation contributes to HNSCC motility. To directly assess this, subsequent Src activation leads to phosphorylation of cortactin cortactin expression in UMSCC2 cells was reduced by siRNAand by Src (13). Because the EGFR-Src pathway is involved in HNSCC expression was restored by transfection of either wild-type FLAG- motility and invasion (38), we sought to determine if down- tagged murine cortactin or with a FLAG-cortactin construct regulation of EGFR activation impairs HNSCC motility and if this containing phenylalanine to tyrosine point mutations at the Src- correlated with reduced cortactin tyrosine phosphorylation. targeted codons 421, 466, and 482 (FLAG-TPM cortactin), HNSCC cells treated with 1 Amol/L of the EGFR small-molecule previously shown to suppress cell motility (12). Overexpression of inhibitor gefitinib were reduced in EGF-stimulated motility by over FLAG-wild type cortactin in UMSCC2 cells without cortactin 80% (excluding the EGFR-null line UMSCC10A; Fig. 6A). Gefitinib knockdown led to a 260% increase in cell migration compared with inhibited motility in a similar but more variable manner when control-transfected cells (Fig. 6D). This increase in motility is likely 10% serum was the chemoattractant, with UMSCC10a displaying due to the additional high amount of recombinant murine 6% inhibition, UMSCC 93%, UMSCC2 77%, UMSCC19 61%, and cortactin expression driven by the cytomegalovirus promoter MSK921 32% (Fig. 6B). Interestingly, the degree of motility present in the recombinant cortactin constructs (data not shown). inhibition by gefitinib in response to EGF and serum is inversely Using these cells as the control for recombinant murine cortactin proportional to the level of cortactin expression in HNSCC (Fig. 6A, expression, expression of FLAG-wild type murine cortactin fol- B versus Fig. 2B). Cortactin immunoprecipitates from HNSCC lines lowing siRNA-mediated knockdown of endogenous cortactin treated with gefitinib and stimulated with 10% serum showed a expression resulted in a 140% reduction in cell migration compared marked reduction in tyrosine phosphorylation in all cases (Fig. 6C), with FLAG-wild type cortactin–overexpressing cells, reflecting the indicating that cortactin tyrosine phosphorylation levels are contribution of endogenous cortactin expression on UMSCC2 modulated by EGFR activation in HNSCC, and that gefitinib- migration. Expression of FLAG-TPM cortactin in UMSCC2 cells mediated inhibition of HNSCC cell migration correlates with down- following cortactin knockdown reduced migration 261% compared regulated cortactin tyrosine phosphorylation. with the overexpression control, 121% compared with cells with The ability of gefinitib to suppress HNSCC motility and cortactin FLAG-wild-type cortactin overexpression in the presence of tyrosine phosphorylation suggested that cortactin tyrosine phos- endogenous cortactin knockdown, and 99% of the transfection www.aacrjournals.org 8021 Cancer Res 2006; 66: (16). August 15, 2006

In agreement with previous work (19, 21), our results show that chromosome 11q13 amplification corresponds with cortactin overexpression in all examined HNSCC cases, correlating with tumor invasion and lymph node metastasis. The enrichment of cortactin within lamellipodia in invasive HNSCC cases is likely reflective of a role in enhancing tumor cell motility, as evidenced by the increased motility and invasion of HNSCC lines with cortactin overexpression. Moreover, in HNSCC cells, the degree of cortactin overexpression corresponds with the level of CTTN amplification, suggesting a direct dose dependency between gene amplification and protein expression. The ability of tumor cells to amplify and tolerate high cortactin levels is to date unique among Arp2/3-activating proteins, possibly due to the lower ability of cortactin to activate Arp2/3 complex compared with N-WASp at similar amounts (15). Slower activation of Arp2/3 by cortactin, even at high cellular concentrations, could still permit the polymerization/depolymerization– based recycling of the lamellipodial actin pool, which is required for proper lamellipodia dynamics and motility (4). In contrast, cells overexpressing activated WASp VCA domains mislocalize Arp2/3 complex and do not form lamellipodia (39), suggesting that overly high WASp protein or activation levels lead to constitutive Arp2/3 activation, which in turn could disrupt lamellipodia actin dynamics to a degree incompatible for promoting cell movement. How cortactin regulates cell motility is becoming increasingly Figure 4. Increased motility and invasion in HNSCC cells with chromosome clearer and more complex. Recent work has shown a role for the 11q3 amplification and cortactin overexpression. HNSCC cells were plated in uncoated transwells (top) or Matrigel-coated wells (bottom) and induced to cortactin amino-terminal acidic domain and actin-binding domains migrate with either 10 ng/mL EGF or 10% FBS for 24 hours. Columns, number of in enhancing and stabilizing protrusive lamellipodia in HT1080 cells that migrated (top) or invaded (bottom) into the lower chamber; bars, SE for fibrosarcoma cells (24), the same regions responsible for acti- a minimum of three independent experiments. P < 0.02 for motility and P < 0.05 for invasion. vation of Arp2/3 complex and stabilization of Arp2/3/F-actin networks (15). The same study also showed decreased G-actin incorporation in the lamellipodia of cells with suppressed control (Fig. 6D). Direct comparison between FLAG-wild type and cortactin expression, consistent with our finding of elevated actin FLAG-TPM cortactin motility in cells with endogenous cortactin incorporation (via the production of free barbed ends) in HNSCC knockdown indicates that FLAG-TPM cells migrate at 45% of the cells with CTTN amplicafication and cortactin overexpression. level of FLAG-wild type cortactin–expressing cells. These data Collectively, these results support a role for cortactin in promoting indicate that cortactin phosphorylation at tyrosines 421, 466, and cell motility through regulating Arp2/3-dependent actin assembly 482 is responsible for 55% of the motility ascribed to cortactin in and/or stabilizing the resulting actin network, resulting increased UMSCC2 cells and confirms that cortactin tyrosine phosphoryla- lamellipodia persistence. Overexpression of cortactin resulting tion is an essential requirement for effective HNSCC motility. from CTTN amplification would likely enhance these effects, as well as carboxyl-terminal SH3 domain–activated cell motility Discussion attributed to increased N-WASp activation (11). Studies to date have shown that cortactin overexpression Concurrent with its role in cell migration, evidence to date resulting from 11q13 amplification in HNSCC and other carcino- indicates that cortactin also regulates tumor cell invasion. In many mas corresponds with poor patient outcome, and that ectopic carcinomas, cortactin is enriched within invadopodia, protrusive overexpression of cortactin in cancer cells lacking 11q13 amplifi- membranous structures in tumor cells that emanate from the cation increases tumor cell motility, invasion, and metastasis. Our ventral cell surface that regulate extracellular matrix degradation results directly show that cortactin overexpression resulting from and protrusion (40). Recent studies have shown that cortactin 11q13 and corresponding CTTN amplification in carcinomas leads is required for invadopodia formation and is essential for the to increased tumor cell motility and invasion. In addition, our data recruitment of membrane type 1 matrix metalloproteinase, the indicate that these effects are likely due to increased binding main enzyme required for invadopodia gelatinase activity, to between cortactin and Arp2/3 complex, potentially serving to invadopodia (41). The ability of cortactin to regulate N-WASp and enhance Arp2/3 actin nucleation activity within tumor cell Arp2/3 activity is likely responsible in part for invadopodia lamellipodia. The resulting enhancement of lamellipodia protru- formation because invadopodia require the activity of both of sion would allow tumor cells to become more motile, increasing these molecules to form and extend (42). In addition to regulating their ability to undergo local-regional invasion, intravasation and invadopodia protrusion, cortactin may also play an important role extravasation, and distant metastasis. The corresponding ability of in the endocytosis of proteolyzed matrix components as evidenced the EGFR inhibitor gefitinib to suppress HNSCC motility and by the requirement of cortactin SH3 domain ligand dynamin 2 for cortactin tyrosine phosphorylation indicates that tyrosine phos- invadopodia-induced matrix degradation (43). phorylation of cortactin may also serve as a valid therapeutic end Although our data indicates that down-regulation of cortactin point marker for metastatic efficacy in the treatment of HNSCC expression with siRNAin tumor cells with CTTN amplification patients with inhibitory compounds targeting the EGFR pathway. significantly decreases carcinoma motility and invasion, it is

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Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2006 American Association for Cancer Research. Cortactin Regulates Tumor Cell Motility and Invasion possible that other genes within the 11q13 amplicon are overex- formation in platelets (45). Pak1 also phosphorylates and activates pressed that may also play a role in regulating cell migration and Arp2/3 complex, inducing lamellipodia extension and cell motility invasion. For example, the locus for the serine/threonine kinase (46). Pak1 may therefore serve to regulate carcinoma motility and Pak1 maps to the 11q13 region, and Pak1 overexpression has been invasion directly through Arp2/3 complex and indirectly through documented in invasive HNSCC and breast carcinoma (44). Pak1 cortactin. Additionally, other genes yet to be characterized within phosphorylates cortactin and is associated with lamellipodia the 11q13 amplicon may also contribute to cell motility. Whereas

Figure 5. Modulation of cortactin expression in HNSCC cells directly affects motility and invasion. A, down-regulation of cortactin in overexpressingHNSCC cells. Top, cells were transfected without RNA (mock), with cortactin siRNA (siRNA), or with a control siRNA conjugated to FITC (FITC-ctl). After 48 hours, cells were lysed and 50 mgtotal cell protein were assayed by Western blot analysis for knockdown of cortactin expression with anticortactin antibodies. Blots were stripped and reprobed with antibodies against h-actin as a loadingcontrol. Bottom, degree of cortactin knockdown in siRNA-treated cells was quantified by densitometry and expressed relative to cortactin levels in cells transfected with FITC-conjugated control siRNA. B, cortactin knockdown in overexpressing HNSCC cells impairs cell motility and invasion. HNSCC cells 48 hours after transfection with cortactin siRNA were assayed for transwell motility (top) and invasion with 10 ng/mL EGF or 10% FBS as mitogenic stimuli (bottom). Columns, percentage motility or invasion compared with each correspondingline transfected with FITC-conjugated control siRNA (control, 100%); bars, SE for a minimum of three independent experiments, *, P < 0.05. C, ectopic cortactin overexpression in HNSCC cells. HNSCC cells with normal cortactin expression (UMSCC8 and HN4) were transfected with GFP-cortactin or GFP control constructs for 24 hours and cell lysates were analyzed by Western blot analysis with an anti-GFP monoclonal antibody (top). Blots were stripped and subsequently probed with cortactin and h-actin antibodies. The positions of GFP-cortactin, GFP, and endogenous cortactin are indicated (right). Bottom, scanned blots were quantified for GFP-cortactin and endogenous cortactin expression, and the degree of total cortactin overexpression is shown relative to GFP transfected controls. D, ectopic cortactin overexpression increases HNSCC motility and invasion. UMSCC8 and HN4 cells expressingGFP or GFP-cortactin were analyzed for motility and invasion as in (B). Columns, percentage motility or invasion compared with each correspondingline transfected with GFP (control, 100%); bars, SE for a minimum of three independent experiments.

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Figure 6. Gefitinib inhibits HNSCC motility and cortactin tyrosine phosphorylation. HNSCC cells were assayed for transwell motility toward 10 ng/mL EGF (A) or 10% FBS (B) in the absence or presence of 1 mmol/L gefitinib for 24 hours (top). Bars, SE for a minimum of three independent experiments. P < 0.05. The percentage motility for gefitinib-treated cells is shown relative to untreated control cells (bottom). C, gefitinib impairs cortactin tyrosine phosphorylation in HNSCC cells. Cortactin immunoprecipitated from HNSCC cells treated without or with 1 mmol/L gefitinib for 4 hours was assayed for tyrosine phosphorylation (pTyr, top) and cortactin expression (bottom) by Western blotting. D, cortactin tyrosine phosphorylation is required for efficient HNSCC migration. UMSCC2 cells were assayed for motility by electric substrate impedance sensingin the presence of 10 ng/mL EGF for 24 hours followingtransfection with control siRNA conjugated to FITC (FITC Control), cortactin siRNA (siRNA), murine FLAG–wild-type cortactin (Cortactin o/x), murine FLAG–wild-type cortactin with cortactin siRNA (Cortactin o/x + siRNA), and murine FLAG-TPM cortactin with cortactin siRNA (Cortactin TPM + siRNA). Columns, percentage motility compared with FITC control-transfected siRNA cells (100%); bars, SE for three independent experiments. *, P < 0.05 compared with FITC control cells.

CTTN may be the predominant 11q13 gene involved in regulating sion (47). The subsequent persistence of EGFR in internalized carcinoma invasion, it is likely that 11q13 amplification exerts a endosomes in HNSCC cells with cortactin overexpression may pleiotropic effect on tumor cell motility and invasion through the provide a sustained counteractive effect to gefitinib, especially in overexpression of several gene products. HNSCC cases with EGFR overexpression, because endosomal EGFR Consistent with previous work (44), gefitinib inhibits motility in is still capable of activating Src (48) and therefore would promote all HNSCC lines analyzed in this study that overexpress EGFR. cortactin phosphorylation. Alternatively, multiple pathways besides Moreover, our data indicate that cortactin tyrosine phosphoryla- EGFR induce cortactin tyrosine phosphorylation (6), potentially tion is down-regulated in HNSCC cells treated with gefitinib, allowing HNSCC cells to circumvent gefitinib-blocked EGFR and indicating that the EGFR-Src signaling pathway is used for Src lead to cortactin tyrosine phosphorylation. The fact that gefitinib activation and subsequent cortactin tyrosine phosphorylation in does not completely abolish cortactin tyrosine phosphorylation in HNSCC. Direct evaluation of cortactin tyrosine phosphorylation on HNSCC (Fig. 6C) supports these hypotheses. HNSCC motility by expression of recombinant phosphorylation– The resistance of serum-stimulated HNSCC cells with cortactin deficient cortactin in cells lacking endogenous cortactin reduced overexpression to the antimotility effects of gefitinib is consistent HNSCC migration levels by 55%, clearly indicating that tyrosine with similar findings in HNSCC cells where cyclin D1–over- phosphorylation of cortactin provides a substantial contribution to expressing lines were resistant to the antiproliferative effects of HNSCC motility. Whereas gefitinib largely inhibited motility (>90%) gefitinib (49). Because cyclin D1 and cortactin are often in response to EGF regardless of 11q13 amplification and cortactin coamplified and overexpressed in HNSCC (18), it is possible that expression status, the ability of gefitinib to inhibit cell migration in the two effects are linked, and that the overexpression of genes response to serum, a more physiologically relevant stimulus, was resulting from chromosome 11q13 amplification in HNSCC and reduced in cells with 11q13 amplification and cortactin over- other carcinomas may have additive effects that lead to gefitinib expression. The HNSCC cell lines used in this study show an resistance. This might partially explain the modest success inverse dose-response relationship between cortactin expression observed in phase II clinical trials with gefitinib in HSNCC (50). levels and cell motility in the presence of gefitinib. One explanation Such results may support validation of cortactin or other genes for this can be derived from a recent study that showed reduced overexpressed from the 11q13 amplicon as potential prognostic ligand-induced EGFR internalization, ubiquitinlyation, and recep- or therapeutic biomarkers for HNSCC and other carcinomas as tor degradation in HNSCC cells containing cortactin overexpres- predictors of tumor cell growth, motility, and invasion.

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Acknowledgments Biology, Vanderbilt University, Nashville, TN) for the N-WASp GST-VCA domain; T. Carey (Department of Otolaryngology, University of Michigan, Ann Arbor, MI) for Received 12/16/2005; revised 4/26/2006; accepted 5/23/2006. UMSCC2, UMSCC8, UMSCC10A, and UMSCC19; and A. Senderowicz (Oral and Grant support: NIH grants DE014364 and DE014578 (S.A. Weed). Pharyngeal Cancer Branch, National Institute of Dental and Craniofacial Research, The costs of publication of this article were defrayed in part by the payment of page Bethesda, MD) for HN4 cell lines; the University of Colorado Cancer Center charges. This article must therefore be hereby marked advertisement in accordance Cytogenetics Facility for related assistance; A. Kapus and C. Di Ciano-Oliveira with 18 U.S.C. Section 1734 solely to indicate this fact. (Department of Surgery, Toronto General Hospital, Toronto, ON, Canada) for help with We thank R. Greer (Department of Diagnostic and Biological Sciences, University the actin incorporation assays; M. Auble (Department of Craniofacial Biology, of Colorado at Denver and Health Sciences Center, Aurora, CO), D. Hines, M. Said, and University of Colorado at Denver and Health Sciences Center, Aurora, CO), T. Cooke K. Shroyer (Department of Pathology, University of Colorado at Denver and Health (A.G. Heinze Inc., Lake Forest, CA), S. Huang (Department of Human Oncology, Sciences Center, Aurora, CO) for tissue archive and pathologic assistance; D. Rimm University of Wisconsin, Madison, WI), J. Altemus (Department of Neurobiology and (Department of Pathology, Yale University, New Haven, CT) for the HNSCC tissue Anatomy, West Virginia University, Morgantown, WV), and S. Frisch (Mary Babb microarray; N. Popescu (Center for Cancer Research, National Cancer Institute, Randolph Cancer Center, West Virginia University, Morgantown, WV) for technical Bethesda, MD) for cortactin P1 genomic probes; A. Weaver (Department of Cancer assistance; and AstraZeneca for gefitinib.

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Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2006 American Association for Cancer Research. Cortactin Overexpression Regulates Actin-Related Protein 2/3 Complex Activity, Motility, and Invasion in Carcinomas with Chromosome 11q13 Amplification

Brian L. Rothschild, Ann H. Shim, Amanda Gatesman Ammer, et al.

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