T-Cadherin Is an Auxiliary Negative Regulator of EGFR Pathway Activity in Cutaneous Squamous Cell Carcinoma: Impact on Cell Motility

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1 OIGINAL ATICLE T-Cdherin Is n Auxiliry Negtive egultor of EGF Pthwy Activity in Cutneous qumous Cell Crcinom: Impct on Cell Motility Emmnouil Kyrikkis 1,4, Kseniy Mslov 1,4, Mri Philippov 1, Dennis Pfff 1, Mnjunth B. Joshi 1, tnislw A. Buechner 2, Pul Erne 3 nd Thérèse J. esink 1 Genetic nd epigenetic studies in different cncers, including cutneous crcinoms, hve implicted T-cdherin (T-cd) s tumor suppressor. Immunohistochemicl nd in vitro studies hve suggested tht T-cd loss promotes incipient invsiveness in cutneous squmous cell crcinom (CC). Moleculr mechnisms re unknown. This study found tht the min consequence of T-cd silencing in CC is fcilittion of lignddependent EGF ctivtion, wheres T-cd overexpression impedes EGF ctivtion. Gin- nd loss-of-function studies in A431 CC cells demonstrte T-cd-controlled responsiveness to EGF with respect to phrmcologicl inhiition of EGF nd to diverse signling nd functionl events of the EGF ctivtion cscde (EGF phosphoryltion, internliztion, nucler trnsloction, cell retrction/de-dhesion, motility, invsion, integrin 1, nd ho smll GTPses such s hoa, c1, nd Cdc42 ctivtion). Further, T-cd modultes the EGF pthwy ctivity y influencing memrne comprtmentliztion of EGF; T-cd upregultion promotes retention of EGF in lipid rfts, wheres T-cd silencing releses EGF from this comprtment, rendering EGF more ccessile to lignd stimultion. This study revels mechnism for fine-tuning of EGF ctivity in CC, wherey T-cd represents n uxiliry negtive regultor of the EGF pthwy, which impcts invsion-ssocited ehviorl responses of CC to EGF. This ction of T-cd in CC my serve s prdigm explining other mlignncies displying concomitnt T-cd loss nd enhnced EGF ctivity. Journl of Investigtive Dermtology (212) 132, ; doi:1.138/jid ; pulished online 17 My 212 INTODUCTION Cutneous squmous cell crcinom (CC), the second most common form of nonmelnom skin cncer, exhiits vrile degrees of typi, invsiveness, nd risk of metstsis (Alm nd tner, 21). Cellulr events prticipting in trnsformtion from noninvsive to invsive nd potentilly metsttic CC re not well understood. trict orchestrtion of cell cell nd cell mtrix dhesion interctions is vitl to pproprite development nd mintennce of ll tissues, including the epidermis (Brker nd McGrth, 21). Tumor cells frequently show deregulted cdherin expression nd inpproprite switching mong fmily memers (Yilmz nd Christofori, 21). Immunohistochemicl nlyses of humn 1 Lortory for ignl Trnsduction, Deprtment of Biomedicine, Bsel University Hospitl, Bsel, witzerlnd; 2 Blumenrin 2, Bsel, witzerlnd nd 3 Division of Crdiology, Kntonsspitl Luzern, Luzern, witzerlnd 4 These uthors contriuted eqully to this work. Correspondence: Thérèse J. esink, Lortory for ignl Trnsduction, Deprtment of Biomedicine, Bsel University Hospitl, ZLF 316 Heelstrsse 2, Bsel CH 431, witzerlnd. E-mil: therese-j.resink@unis.ch Arevitions: A431, humn epidermoid CC cell line; EMT, epithelil-tomesenchyml trnsition; p-, phosphorylted; CC, squmous cell crcinom; st, T-cd-silenced cell; T þ, T-cd-overexpressing cell; T-cd, T-cdherin eceived 2 Decemer 211; revised 16 Ferury 212; ccepted 1 Mrch 212; pulished online 17 My 212 cutneous CC specimens hve identified gin of N-cdherin (Nguyen et l., 211) nd desmoglein 2 (Kurzen et l., 23), nd loss of E-cdherin (Lykhovitsky et l., 24) nd glycosylphosphtidylinositol-nchored T-cdherin (T-cd) (Tkeuchi et l., 22; Pfff et l., 21) s cndidte prticipnts in the development of incipient invsive CC. Knowledge regrding moleculr mechnisms wherey T- cd prticiptes in CC progression is scnt. Loss of T-cd expression through llelic loss or errnt gene methyltion in invsive cutneous CC ws reported (Tkeuchi et l., 22). Anlysis of T-cd expression in reltion to histologicl clssifiction of degree of differentition reveled tht T-cd loss in cutneous CC ws not uiquitous ut occurred loclly in ssocition with histologicl fetures of potentilly more mlignnt nd invsive phenotype (Pfff et l., 21). In vitro studies using HC-1 or A431 CC cell lines collectively demonstrted tht T-cd silencing incresed migrtory, invsive, nd prolifertion potentil, wheres the converse ws true for T-cd overexpression (Mukoym et l., 25; Pfff et l., 21, 211). However, in murine xenogrft model, either gin or loss of T-cd in A431 incresed tumor expnsion in vivo (Pfff et l., 211). This prdox ws ttriuted to enhnced vsculr endothelil cell growth fctor medited enhncement of ngio/lymphngiogenesis for T-cd overexpression, nd to enhnced & 212 The ociety for Investigtive Dermtology

2 EGF/extrcellulr signl regulted kinse-medited prolifertion for T-cd silencing. Potentil linkge etween EGF nd T-cd wrrnts ttention. More thn 9% of CCs express elevted levels of EGF. EGF hs crucil role in CC prolifertion, invsion, nd metstsis, nd gthering evidence suggests tht EGF-directed therpies my e useful djuncts to surgicl tretment for CC (Fung nd Grndis, 21). Understnding mechnisms underlying enhnced EGF expression nd/or ctivity is importnt for optimiztion of EGF-directed therpies. This study investigted whether nd how T-cd expression impcts invsive nd motogenic responses of A431 to EGF. We report tht T-cd modultes responsiveness of the EGF pthwy to EGF y influencing memrne comprtmentliztion of the receptor. ilencing or overexpression of T-cd, respectively, fcilittes or impedes lignd-dependent EGF ctivtion, with similr significnce for dhesion, motility, nd invsion responses of CC to EGF. EULT Ectopic ltertions of T-cd expression in A431 cells re not ccompnied y chnges in mrkers of EMT ut lter sensitivity of cells to EGF inhiitors This study used CC cell line A431 stly trnsduced with respect to either T-cd overexpression (T þ ; using plvx-puro vector crrying humn T-cd complementry DNA) or T-cd silencing (st; using plko.1-puro vector crrying T-cd short hirpin NA), nd stly trnsduced with empty plvx-puro vector (E) or plko.1-puro vector crrying rndom short hirpin NA (st) s the respective controls (Pfff et l., 21). upplementry Figure 1 online shows levels of T-cd protein nd trnscript expression in the trnsductnts. We previously reported tht motile nd invsive cpcities of T þ nd st were decresed nd incresed, respectively (Pfff et l., 21), ut did not investigte mechnisms underlying these phenomen. The occurrence of epithelil-to-mesenchyml trnsition (EMT) is commonly ssocited with migrtion nd invsion of tumor cells (Yilmz nd Christofori, 21). Immunofluorescence microscopy (upplementry Figure 2 online), immunolotting (Pfff et l., 21), nd quntittive PC nlysis (dt not shown) reveled no influence of T-cd expression on either levels or cellulr distriution of E- cdherin, P-cdherin, or vimentin. N-cdherin ws not detectle with ny nlyticl method pplied (dt not shown). As T-cd silencing in norml kertinocytes lso increses migrtory-invsive cpcities nd fils to influence epithelil nd mesenchyml mrker levels (Pfff et l., 21), loss of T-cd in CC likely ffects migrtion nd invsive ehvior y mechnisms different from those typiclly ssocited with EMT (Yilmz nd Christofori, 21). As n lterntive to EMT-sed mechnisms, we considered whether effects of T-cd on A431 motile-invsive ehvior involve EGF pthwy ctivity. A431 expresses high levels of EGF, which, lthough mking cell prolifertion less dependent on exogenous EGF (Fn et l., 1994), enhnces EGF-induced motogenic responses (Mlliri et l., 1998). Assys of three-dimensionl spheroid invsion (Figure 1) nd of trnswell migrtion (upplementry Figure 3 online) confirmed (Pfff et l., 21) decresed nd incresed invsiveness, respectively, for T þ nd st, nd further demonstrted tht invsion (Figure 1) nd trnsmigrtion (upplementry Figure 3 online) y T þ ws less sensitive thn st to EGF tyrosine kinse inhiitors gefitini or lptini. These dt infer EGF pthwy inhiition in T þ, nd rise the possiility tht T-cd expression levels regulte EGF-dependent motile-invsive responses in A431. T-cd expression levels modulte EGF ctivity nd internliztion To determine whether T-cd expression might influence EGF ctivity, we exmined indices of EGF ctivtion t the level of EGF itself, nmely EGF phosphoryltion nd internliztion (chlessinger, 2; orkin nd Goh, 29). T-cd overexpression in the CC cell line HC-1 ws reported to result in constitutive reduction of totl nd phosphorylted EGF (p-egf) levels (Mukoym et l., 27). We found tht neither T-cd overexpression nor T-cd silencing in A431 ffected constitutive levels of totl or p-egf (Figure 1c). However, upon EGF stimultion st exhiited more prompt nd mplified EGF phosphoryltion, wheres this ws lunted in T þ (Figure 1c). EGF-induced loss of totl EGF occurred fster in st nd ws delyed in T þ (Figure 1c). We next pplied n immunofluorescencesed method (Frncvill et l., 29) tht llows monitoring of receptor internliztion. As expected, EGF stining t cell cell contcts ws prominent t seline nd wekened upon EGF stimultion in ssocition with incresed intrcellulr stining (Figure 2, upplementry Figure 4 online, no cid wsh). EGF-induced internliztion occurred fster in st nd ws delyed in T þ (Figure 2, upplementry Figure 4 online, with cid wsh). Even t seline, st exhiited greter intrcellulr stining thn T þ. To otin iochemicl evidence for differences in plsm memrne-to-intrcellulr trnsloction of EGF, we performed immunolotting using lystes from cells collected without trypsiniztion (totl EGF) or fter trypsiniztion (internlized EGF). Intrcellulr EGF levels under sl nd EGF-stimulted conditions were elevted in st nd lowered in T þ (Figure 2). Nucler EGF nd p-egf levels were exmined y immunolotting of nucler extrcts. Purity of nucler extrcts ws checked y immunolotting for nucler mrker lmin A/C (positive), plsm memrne mrkers E-cdherin nd ZO-1 (negtive), nd cytoplsm mrkers glycerldehyde-3-phosphte dehydrogense nd Grp78 (negtive) (upplementry Figure 5 online). At seline, EGF nd p-egf levels were higher in st with trend towrd decrese in T þ (Figure 2c). Upon EGF stimultion, nucler EGF nd p-egf levels incresed in ll trnsductnts, ut the increse ws ugmented for st nd lunted for T þ (Figure 2c). Thus, T-cd silencing or T-cd overexpression, respectively, fcilittes or impedes EGF ctivtion. T-cd promotes retention of EGF in lipid rft domins Chnges in EGF memrne comprtmentliztion hve mjor role in its ctivtion: in the sl stte EGF prtitions 2276 Journl of Investigtive Dermtology (212), Volume 132

3 Invded re fter 3 dys (vs. E or sc control) Invded re fter 3 dys (vs. untreted controls) Control Lptini (1 μm) E Gefitini (1 μm) lptini s lptini gefitini s gefitini EGF inhiitor (μm) Control Lptini (1 μm) Gefitini (1 μm) sc st c p-egf (vs. E or sc time) Totl EGF (vs. E or sc time) E E E E E E p-egf (17 kd) EGF (17 kd) GAPDH (37 kd) sc st sc st sc st sc st sc st sc st p-egf (17 kd) EGF (17 kd) GAPDH (37 kd) Time (min) Time (min) Figure 1. T-cd expression levels in A431 lter sensitivity to EGF inhiition nd EGF ctivity. (, ) Mtrigel-emedded spheroids were cultured for 3 dys without (control) or with inclusion of lptini or gefitini (1 mm in, 1mM in ). () epresenttive imges of the spheroids re shown (r, 2 mm pplicle to ll imges), Po.1. () Po.1 compres T þ nd st for either gefitini or lptini. (c) Cells were stimulted with EGF (1 ng ml 1 ) for the indicted times. Whole-cell lystes were nlyzed for p-egf nd totl EGF y immunolotting. GAPDH served s internl loding control. Altertions in levels in E or T þ nd sc or st re expressed reltive to levels in E nd sc controls, respectively. P t lest o.1. E, empty vector cell; GAPDH, glycerldehyde-3-phosphte dehydrogense; p-egf, phosphorylted EGF; sc, non-trget short hirpin NA cell; st, T-cd-silenced cell; T þ, T-cdoverexpressing cell; T-cd, T-cdherin. into lipid rft domins of the plsm memrne, nd sequestrtion of EGF within the rfts exerts inhiitory effects on EGF signling (Pike, 25; Blis nd Posner, 21). As T-cd is lso resident within lipid rft domins (Philippov et l., 1998), we considered whether its expression level might influence EGF locliztion nd/or retention. Confocl microscopy imges of empty vector nd non-trget short hirpin NA trnsduced cells doule-stined for EGF/T-cd depicted similr leling ptterns, with prominent co-stining t cell cell contcts (Figure 3). Co-stining signl intensities were enhnced in T þ nd negligile in st (Figure 3). Isoltion of memrne rfts using detergent nd nondetergent protocols (Figure 3 nd c) reveled higher nd lesser co-frctiontion of EGF in T-cd- nd cveolin-1- enriched, clthrin-negtive rft domins from T þ nd st, respectively. eciprocl co-immunoprecipittion experiments using vriety of detergent-sed lysis uffer conditions (upplementry informtion online) filed to revel co-precipittion of EGF with T-cd (upplementry Figure 6 online). To ensure tht our uffer conditions mintin protein - protein interctions tht occur within lipid rfts, we proed nti-egf immunoprecipittes for the presence of upa (upplementry Figure 6 online), rft-ssocited protein (tuch nd Hnisch, 211) reported to complex with EGF (Liu et l., 22; Mzzieri et l., 26). We hve previously demonstrted ssocition of T-cd with Grp78 nd integrin-linked kinse in endothelil cells (Joshi et l., 27; Philippov et l., 28) ut found no such ssocitions in A431 (dt not shown), suggesting tht T-cd my hve different memrne prtners in different cell types. However, we reconfirmed formtion of T-cd Grp78 nd T-cd IL-8 complexes in endothelil cells (upplementry Figure 7 online), thus vlidting our uffer conditions lso in the cse of T-cd. Thus, EGF nd T-cd co-loclize nd T-cd expression levels modulte retention of EGF in rft domins, ut EGF nd T-cd pper not to e physiclly ssocited. T-cd expression levels influence EGF-induced chnges in cell retrction nd motility etrctile nd motile responses to EGF were exmined using time-lpse videomicroscopy nd morphometric nlysis. As expected (Chinkers et l., 1981), EGF induces rpid cell rounding nd retrction from the sustrte (Figure 4). st exhiited more pronounced rounding/retrction, wheres the response ws reduced in T þ (Figure 4). upplementry videos 1 nd 3 online illustrte distinct retrction cpcities of st nd T þ. TITC-phlloidin stining of cells

4 No cid wsh With cid wsh No cid wsh With cid wsh E sc st EGF (1 min) +/ Trypsin EGF levels (vs. E ) EGF levels (vs. sc ) 1..5 EGF GAPDH 1..5 EGF GAPDH EGF (3 min) + + c EGF (3 min) E E E E sc st sc st sc st sc st EGF (vs. E or sc time) p-egf (vs. E or sc time) EGF (3 min) E E sc st sc st EGF p-egf Lmin A/C EGF p-egf Lmin A/C Figure 2. T-cd expression levels modulte EGF internliztion. () Alex Fluor 488 IgG ws used to visulize internlized EGF (with cid wsh) nd totl EGF (no cid wsh) fter live-cell incution with nti-egf ntiodies (r, 2 mm for ll photomicrogrphs). () Immunolotting for EGF using lystes from cells hrvested without trypsiniztion (totl EGF) or fter trypsiniztion (internlized EGF). (c) Immunolotting for EGF in nucler extrcts. EGF nd p-egf levels in T þ nd st re expressed reltive to those in their respective E nd sc controls. Po.5, Po.1. E, empty vector cell; GAPDH, glycerldehyde-3-phosphte dehydrogense; p-egf, phosphorylted EGF; sc, non-trget short hirpin NA cell; st, T-cd-silenced cell; T þ, T-cd-overexpressing cell; T-cd, T-cdherin. cultured on geltin-coted glss coverslips showed prtil rettchment to the sustrtum following prolonged (24 hours) EGF stimultion. st exhiited retrcted, poorly spred morphology, nd extensions were thred-like nd spiky, superficilly resemling neurite-like protrusions, wheres protrusions in T þ were more rod nd flttened (upplementry Figure 8 online). To monitor the effects of T-cd on motility responses to EGF, cells were seeded t lower density to minimize cell clustering. EGF ws dded to the medium (Figure 4) or presented within drop of polymerized firin (Figure 4c). In oth cses, movement speeds were incresed nd decresed in st nd T þ, respectively. With EGF dded to the medium, cell movement ws ssocited with dynmic extension nd retrction of lmellipodi nd filopodi, nd, in ccordnce with sence of chemotctic grdient, the direction of motility ws rndom (Figure 4). With firin-emedded EGF, cells moved towrd the source of EGF nd with moeoid morphodynmics (Figure 4c). T-cd expression levels influence sl nd EGF-induced chnges in the ctivity of integrin 1 nd ho smll GTPses To investigte the moleculr mechnisms underlying T-cd effects on cell retrction nd motility, we ddressed ctivity levels of integrin 1 nd the ho fmily of smll GTPses, which re crucil regultors of cell dhesion nd migrtion (Hood nd Cheresh, 22; Lozno et l., 23). HUT-4 ntiodies, which specificlly recognize the ctive conformtion of 1 integrins (Monghn-Benson nd McKeown- Longo, 26), were used to mesure the levels of ctivted integrin 1 on the cell surfce. Under norml culture conditions, st contined higher levels of totl integrin 1 nd ound less HUT-4 (Figure 5). There were trends towrd reduced totl integrin 1 nd incresed HUT-4 inding in T þ. Expression of dt s n ctivity rtio (ctive integrin 1/totl integrin 1) reveled significntly incresed integrin 1 ctivtion in T þ nd confirmed the decresed integrin 1 ctivtion in st. EGF-induced integrin 1 ctivtion ws incresed in T þ ut decresed in st (Figure 5). EGF-induced ctivtion of c1 nd Cdc42 ws incresed in T þ ut decresed in st (Figure 6 nd ). Conversely, hoa ctivtion ws decresed in T þ nd incresed in st (Figure 6c). EGF-induced phosphoryltion of 2-kD myosin light chin (MLC 2 ) on threonine 2/serine 19, medited y OCK downstrem effector of ho, ws higher in st (Figure 6d). To confirm the involvement of hoa in T-cd-dependent effects on cell retrction, st nd T þ were co-trnsduced with denovector expressing dominntnegtive mutnt hoa (Adv-N19hoA). Videomicroscopy (upplementry videos 1, 2, 3 nd 4 online) nd nlysis 2278 Journl of Investigtive Dermtology (212), Volume 132

5 T-cd EGF Hoechst Merged E sc st (Detergent isoltion) c (Non-detergent isoltion) EGF level (vs. E or sc) trting lyste () fts frction () Non-rfts frction (N) EGF (17 kd) T-cd (15/13 kd) Cveolin1 (22 kd) Clthrin (18 kd) N N N N N N N N Figure 3. Co-locliztion of T-cd nd EGF. () Fixed nd permeilized cells were co-stined for T-cd (red), EGF (green) with nucler counterstining using Hoechst (lue), nd nlyzed using n LM-71 lser scnning microscope. epresenttive imges of single nd merged stinings re shown (r, 2 mm pplicle to ll photomicrogrphs). ft domins were isolted nd frctionted using detergent- (Triton X-1; ) nd non-detergent- (Optiprep; c) sed protocols. trting lystes (), rft (), nd ottom non-rft (N) frctions were nlyzed y immunolotting for EGF, T-cd, cveolin 1, nd clthrin hevy chin. Trnsduction sets (seprted with white lines) were run on the sme gel; representtive lots re shown. EGF levels in isolted from T þ or st cells re expressed reltive to those in their respective E or sc controls. Po.1, Po.1. E, empty vector cell; sc, non-trget short hirpin NA cell; st, T-cd-silenced cell; T þ, T-cd-overexpressing cell; T-cd, T-cdherin. of surfce re coverge (Figure 6e) showed tht N19hoA reduced the enhnced retrction of st. DICUION This study hs identified T-cd s negtive uxiliry regultor of EGF pthwy ctivtion in CC, with consequences for functionl responses of CC to EGF. First, spheroid invsion ssy reveled tht sts were highly sensitive to inhiition y EGF inhiitors lptini nd gefitini, indicting the contriution of EGF pthwy ctivity to their incresed invsive potentil. econd, greter responsiveness to EGF in st ws evident for mny signling nd functionl events of the EGF ctivtion cscde (e.g., EGF phosphoryltion, internliztion, nucler trnsloction, cell retrction/de-dhesion). The converse stte of ttenuted responsiveness to EGF ws true for T þ. These dt might e interpreted to suggest tht when T-cd is present in the cell memrne it cts s rke on EGF signling, wheres loss of T-cd fcilittes EGF responsiveness nd pthwy ctivtion. We explored mechnisms underlying T-cd-dependent regultion of EGF ctivity. Confocl microscopy reveled

6 Δ urfce re (mm 2 ) 2, 1,5 1, 5 5 No EGF + EGF Δ urfce re (15 min) st peed of movement (mm min 1 vs. E or sc) E sc st.5 mm c peed of movement (mm min 1 vs. E or sc) E sc st Figure 4. T-cd expression influences EGF-induced chnges in cell retrction nd motility. () Chnge (D) in surfce re ws clculted s the difference in cluster res efore nd fter EGF stimultion (1 ng ml 1 ), s illustrted in still-frmes cquired from T þ nd st. Dot plots present dt for ech cluster nlyzed, with medin nd interqurtile rnge; Po.5, Po.1. (, c) EGF ws included in medium (1 ng ml 1 ; ) or presented within centrlly positioned firin plug (7 ml contining 2 ng EGF per ml; c). Movement speeds (mm min 1 )oftþ or st re expressed reltive to speeds of their E (.44±.12 for ;.5±.12 for c) or sc (.39±.12 for ;.43±.11 for c) controls; Po.5, Po.1. Imges in nd c re still-frmes (rs, 5 mm) extrcted from videos of st. E, empty vector cell; sc, non-trget short hirpin NA cell; st, T-cd-silenced cell; T þ, T-cd-overexpressing cell; T-cd, T-cdherin. co-locliztion etween T-cd nd EGF. However, sed on our filed ttempts to co-precipitte T-cd nd EGF from the plsm memrne, direct interction etween the two proteins would seem unlikely. Another possiility is tht T-cd influences memrne distriution of EGF. everl studies (reviewed in Pike, 25; Blis nd Posner, 21) hve demonstrted tht EGF ctivity depends on its locliztion within lipid rfts, which re specilized domins of the plsm memrne tht re enriched in sphingolipids nd sterols nd ct s ssemly pltforms for receptors nd their prtner signling molecules. ft disruption resulted in loss of IgCAM-medited EGF ctivtion in the olfctory system (Gison et l., 29), suggesting tht intctness of lipid rft pltforms is prerequisite for EGF function. On the other hnd, evidence lso supports the fct tht lipid rfts negtively regulte EGF ctivity y inhiiting lterl movement of EGF nd decresing proility of EGF dimeriztion. EGF ws inctivted when trpped in rft comprtments (Mineo et l., 1999), wheres inding of cognte lignds to EGF cused its relese out of lipid rfts, dimeriztion, nd stimultion of tyrosine kinse ctivity (Lmert et l., 26). ft destruction cused EGF-dependent hyperctivtion of Erk1/2 (Furuchi nd Anderson, 1998). It lso induced spontneous ctivtion of EGF even in the sence of lignd y cusing its relese into smll confined domins of the plsm memrne where the receptors dimerize nd utophosphorylte their cytoplsmic domins owing to their high density nd/or seprtion from inhiitory molecules tht ssocite with the receptors in rfts (Pike nd Csey, 22; ingerike et l., 22; Lmert et l., 26). Our nlysis of EGF levels in lipid rft domins provides evidence tht T-cd upregultion in A431 leds to retention 228 Journl of Investigtive Dermtology (212), Volume 132

7 Integrin β1 GAPDH Integrin β1 levels (vs. E or sc control) tio ctive β1/totl β1 (vs. E or sc control) Totl β1 Active β1 (13 kd) (218 kd) Totl β1 Active β1 Integrin β1 levels (vs. E or sc control) tio ctive β1/totl β1 (vs. E or sc control) Totl β1 Active β1 GAPDH EGF Totl β1 Assy control +Mn 2+ Active β1 EGF EGF Figure 5. T-cd expression ffects constitutive integrin 1 ctivity nd EGF-induced integrin 1 ctivtion. Integrin ctivtion under norml culture conditions () or following EGF stimultion (1 ng ml 1, 15 minutes; ) ws mesured using the HUT-4 ntiody specific for ctivted integrin 1. Cells were exposed to MnCl 2 (5 mm) s positive control. Typicl lots re shown. The higher mss of ctive integrin 1 (218 kd) derives from the complex of integrin 1 (13 kd) nd HUT-4 ntiody (88 kd). Totl nd ctive integrin 1 levels re presented individully or s rtio (ctive 1/totl 1), nd chnges in T þ or st re expressed reltive to vlues in their respective E nd sc controls; Po.5, Po.1, Po.1. E, empty vector cell; GAPDH, glycerldehyde-3-phosphte dehydrogense; sc, non-trget short hirpin NA cell; st, T-cd-silenced cell; T þ, T-cd-overexpressing cell; T-cd, T-cdherin. of EGF in lipid rfts, wheres T-cd silencing releses EGF from this comprtment. This, tken together with the differentil EGF responsiveness of T þ (decresed) nd st (incresed) would e concordnt with the view tht lipid rft locliztion negtively regultes EGF ctivity. As it is T-cd downregultion/loss tht occurs in vivo in invsive CC tumor specimens (Tkeuchi et l., 22; Pfff et l., 21), centrl issue of the study concerns consequences of T-cd loss on EGF-dependent modultion of signling systems controlling CC migrtion nd invsion. We found tht st exhiited incresed totl levels of integrin 1, wheres their integrin 1 ctivtion sttus (ctive/totl integrin 1 rtio) ws decresed. The ltter is consistent with incresed EGF ctivity in st, which my promote integrin 1 internliztion (Mukoym et l., 27) nd therefore inctivtion. Mny studies demonstrte ssocitions etween regultion of integrin expression nd cncer. Chnges in integrin ptterns llow recognition of vrile mtrices y cncer cells nd modulte signling nd gene expression (Hood nd Cheresh, 22). It is widely held tht integrin 1 is required for cncer cell motility nd invsion. Therefore, the finding tht st (which re chrcterized y enhnced invsive potentil) disply decresed integrin 1 ctivity ws somewht unexpected. However, CC cncers re heterogeneous nd my exhiit vrile ltertions in integrin expression nd function (Jnes nd Wtt, 26). Integrin 1 is overexpressed in cervicl CC (Hughes et l., 1994) nd vulvr CC prticulrly t the invding tumor orders (Brocknk et l., 25). In contrst, reduced expression of integrin 1 ws reported in orl CC (Jones et l., 1993; Bgutti et l., 1998). Lower expression of integrin 1 t the invsive front of orl CC correlted with poor prognosis (Ohr et l., 29). There re lso some miguities with respect to prticiption of integrin 1 in EGF-modulted CC growth nd differentition. Forced expression of integrin 1 in the suprsl lyer of the epidermis suppresses cell differentition nd cell cycle exit (Crroll et l., 1995), possily y promoting Erk1/2 ctivity (Zhu et l., 1999). In contrst, in orl CC cell line CC4, which expresses n inctive mutnt integrin 1 nd is poorly differentited, the introduction of wild-type integrin 1 stimultes differentition (Evns et l., 23), wheres integrin 1 knockdown in A431 fils to ffect either prolifertion or Erk1/2 ctivity in vitro or in vivo (Brocknk et l., 25). Decresed sl ctivity nd EGF-dependent inctivtion of integrin 1 in st might e relted to two phenomen. First, initition of CC motility y EGF involves cell morphology chnges including cytoskeleton rerrngement, remodeling of cell mtrix contcts, loss of stress fiers, induction of

8 Active c1 (vs. E or sc) 1..5 Active Cdc42 (vs. E or sc) c Active hoa (vs. E or sc) c1 GTP (21 kd) Cdc42 GTP (22 kd) Totl hoa (22 kd) GT PBD (26 kd) GT PBD (26 kd) GAPDH (37 kd) d p-mlc 2 levels (vs. own time) 4 E 3 sc st 2 1 p-mlc 2 (2 kd) Time (min) e Degree of retrction fter 15 min (% chnge in surfce coverge) st st GAPDH (37 kd) Adv-E Adv-N19hoA Figure 6. T-cd expression ffects EGF-dependent ctivtion of smll ho GTPses nd myosin light-chin phosphoryltion. ( c) Trnsductnts were stimulted with EGF (1 ng ml 1 ) for 3 minutes ( c), 5 15 minutes (d), nd 15 minutes (e). Activtion of smll GTPses c1 () nd Cdc42 () were mesured y pull-down ssy. Activtion of hoa (c) ws mesured y G-LIA. Activity levels in T þ or st re expressed reltive to those for E or sc controls. (d) Immunolotting for p-mlc 2.(e) Time-lpse videomicroscopy (1 frme per 5 minutes) ws performed on T þ nd st co-trnsduced with control empty denovector (Adv-E) or denovector expressing dominnt-negtive hoa (Adv-N19hoA). etrctile response ws mesured s percentge chnge in totl surfce re occuption. Illustrtive videos nd legends re provided in upplementry informtion online; Po.5, Po.1, Po.1. E, empty vector; GAPDH, glycerldehyde-3-phosphte dehydrogense; GT PBD, glutthione -trnsferse protein inding domin fusion protein; GTP, gunosine triphosphte; sc, non-trget short hirpin NA cell; st, T-cd-silenced cell; T þ, T-cd-overexpressing cell; T-cd, T-cdherin. corticl ctin polymeriztion, nd cell rounding. Although integrins my e needed for lter cell motility, the initil response to EGF is ssocited with loss of cell mtrix ttchments (Huck et l., 21). The fct tht st exhiit pronounced EGF-induced integrin 1 inctivtion together with rpid disssemly of cell mtrix contcts, cell retrction, nd rounding my reflect generlly enhnced EGF ctivtion sttus. Decresed sl integrin 1 ctivity in st might e explined y residul EGF ctivity even in the sence of high concentrtions of exogenous EGF, invoking possiility tht in vivo T-cd loss my contriute to oth lignd-dependent nd -independent EGF ctivtion. econd, decresed integrin 1 ctivity my e ssocited with specific type of cell motility. ingle cncer cells my move using t lest two distinct migrtion modes, nmely mesenchyml or moeoid migrtion (Lmmermnn nd ixt, 29; Ymzki et l., 29; Prri nd Chirugi, 21; Yilmz nd Christofori, 21). Mesenchyml migrtion is chrcterized y elongted morphology, cler front-to-rer end polriztion, well-formed lmellipodi, dependence on integrins nd mtrix metlloproteinses, nd incresed c1 signling. Cells moving in n moeoid mnner exhiit rounded morphology with le-like protrusions, decresed dhesion to the surfce, integrin inctivtion, nd concomitnt chnges in smll GTPses ctivity, nmely, c1 inhiition nd hoa/ock ctivtion. In ccordnce with literture (Mlliri et l., 1998; hi, 27), our videomicroscopy experiments demonstrte tht A431 my migrte in n moeoid mnner. Furthermore, the nture of chnges in ctivities of integrin 1 (decresed) nd smll GTPses (c1 decresed, hoa incresed) in st is precisely tht expected for enhnced moeoid motility (Lmmermnn nd ixt, 29; Ymzki et l., 29; Prri nd Chirugi, 21). In vivo, such ltertions in cell phenotype nd integrindependent dhesion my contriute to more ctive migrtion nd chemotxis, leding to incresed tumor cell dissemintion (Kren et l., 27). Literture evidence supports the existence of rod spectrum of memrne molecules cting s uxiliry regultors ( co-receptors ) of growth fctor receptor ctivity (Kirkride et l., 25). Fine-tuning of receptor ctivtion is often chieved y their rpid redistriution into vrious plsm memrne domins or internliztion, llowing fst ssocition of the receptor with vrious scffolds nd dptors or signl ttenution. This enles prompt cell rections to the chnging environment, which is prticulrly importnt, for 2282 Journl of Investigtive Dermtology (212), Volume 132

9 exmple, for directed migrtion during ngiogenesis, neurite outgrowth, or tumor front invsion. Exmples of such pired regultion re interply etween neurl cell dhesion molecule NCAM nd firolst growth fctor receptor FGF-1 in tumor dissemintion (Frncvill et l., 29; Zecchini et l., 211), IgCAM-medited EGF ctivtion in the developing olfctory pthwy (Gison et l., 29), nd others (Kirkride et l., 25). Mny of these co-receptors, such s T-cd, re surfce dhesion molecules loclizing in lipid rfts tht link signling components together nd define the mechnism of receptor complex ctivtion, stiliztion, or internliztion. We invoke T-cd s further exmple of co-receptor involved in sptil nd temporl djustment of cell sensitivity to growth nd chemotctic signls. pecific moleculr mechnisms of T-cd-dependent regultion of EGF comprtmentliztion nd its consequences (e.g., lignd inding, receptor dimeriztion, interction with dptor proteins) re yet to e elucidted. In conclusion, loss of T-cd in CC my led to lignddependent EGF hyperctivtion with resultnt exggertion of invsive nd ggressive tumor ehvior. Epigenetic studies (reviewed in Lochter et l., 1997) hve reported silencing of T-cd gene CDH13 lso in melnoms nd other mlignncies (e.g., rest, pncretic, lung, ovrin, inter li) in which enhnced EGF ctivity frequently occurs. Therefore, it is pertinent to consider whether suppression of EGF pthwy ctivtion might e common tumor suppressor mechnism of T-cd. MATEIAL AND METHOD Cell culture Lentivector-medited genertion of A431 (epidermoid crcinom of skin; ATTC (Mnsss, VA), CL-1555) stly trnsduced with respect to T-cd overexpression (T þ ) or T-cd deficiency (st), nd with empty vector (E) or non-trget short hirpin NA (sc) cells s respective controls, hs een descried (Pfff et l., 21). Cells were normlly cultured in DMEM contining 1% fetl clf serum (DMEM/fetl clf serum) nd were serum deprived culture for 4 hours in DMEM contining.1% BA (DMEM/BA) efore EGF stimultion (1 ng ml 1 ). Invsion nd trnsmigrtion ssys pheroid invsion nd trnswell migrtion ssys were performed s descried (Pfff et l., 21), with minor modifictions (upplementry informtion online). Fluorescence microscopy Fluorescence microscopy techniques hve een descried (Philippov et l., 23, 25; Pfff et l., 21). tining protocols, ntiody sources, nd informtion on microscopes, cmers, nd cquisition softwre re detiled in upplementry informtion online. Time-lpse videomicroscopy nlysis of cell surfce re nd cell motility Time-lpse videomicroscopy techniques hve een detiled (Kyrikkis et l., 21). eeding densities, cquisition, nd morphometric nlyticl methods re detiled within upplementry informtion online. Imges were cquired t rte of 1 frme per 5 minutes for 15 minutes to record erly retrction/spreding or 1 frme per 5 or 1 minutes for hours to record motility. Assys for EGF internliztion nd trnsloction EGF internliztion ws monitored using previously descried live-stining immunofluorescence-sed method (Frncvill et l., 29) with minor modifictions (upplementry informtion online). Mouse nti-egf IgG (A-5, clone H11) nd nti-mouse Alex Fluor 488 IgG were used for EGF detection. Plsm memrne to-intrcellulr trnsloction of EGF ws determined using conventionl nucler frctiontion nd monolyer trypsiniztion pproches (upplementry informtion online). Lipid rfts isoltion Lipid rfts were isolted following exct protocols descried for detergent (Triton X-1)-sed isoltion/frctiontion through 5 3% sucrose grdient (Philippov et l., 1998) nd non-detergentsed isoltion/frctiontion through grdient of 2%-Optiprep (Nycomed Phrm A, Oslo, Norwy; Mcdonld nd Pike, 25). Assys for ctivity of ho smll GTPses Pull-down ssy for quntifiction of ctive gunosine triphosphteound c1 nd Cdc42 ws performed s descried (en nd chwrtz, 2), with minor modifictions (upplementry informtion online). hoa GTPse ctivity ws determined using the G-LIA hoa Activtion Assy Kit (Cytoskeleton, Denver, CO). Mesurement of the occupied, ctive conformtion of integrin 1 Activted integrin 1 ws detected using HUT-4 mas s descried (Monghn-Benson nd McKeown-Longo, 26), with minor modifictions (upplementry informtion online). Immunolotting D-PAGE nd immunolotting protocols hve een detiled (Kuzmenko et l., 1998). Primry ntiodies ginst the following proteins were used: T-cd, cveolin 1, clthrin hevy chin, hoa, c1, Cdc42, EGF, p-egf (Tyr168), p-mlc (Thr18/er19) 2, humn integrin 1, glutthione -trnsferse, ZO-1, lmin A/C, nd glycerldehyde-3-phosphte dehydrogense. ources of primry nd secondry ntiodies re given in upplementry informtion online. epresenttive lots re shown. ttisticl nlysis All experiments were conducted on t lest three seprte occsions, nd unless otherwise stted ll results re given s men±d. Differences were determined using one-wy repeted mesures nlysis of vrince with Tukey s multiple comprison using the GrphPd Prism 5. softwre (GrphPd oftwre, n Diego, CA). Po.5 ws considered significnt. CONFLICT OF INTEET The uthors stte no conflict of interest. ACKNOWLEDGMENT This work ws supported y Kresforschung chweiz (grnt no. KF ), Herzkreisluf tiftung, nd wisslife Juilümsstiftung

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