Axl Promotes Cutaneous Squamous Cell Carcinoma Survival through Negative Regulation of Pro-Apoptotic Bcl-2 Family Members

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1 ORIGINAL ARTICLE Axl Promotes Cutneous Squmous Cell Crcinom Survivl through Negtive Regultion of Pro-Apoptotic Bcl-2 Fmily Memers Emmnouil S. Ppdkis 1, Monik A. Cichoń 1, Jshmin J. Vys 1, Nkul Ptel 1, Lucy Ghli 2, Rino Cerio 1, Aln Storey 3 nd Edel A. O Toole 1 Expression of Axl, receptor tyrosine kinse, is incresed in cutneous squmous cell crcinom (SCC). Exmintion of series of cutneous SCC tumors reveled positive phospho-akt (P-Akt) stining ccompnied y wek TUNEL stining in Axl-positive tumors, suggesting n nti-poptotic role for Axl in SCC survivl. The role of Axl in -induced poptosis ws investigted in cutneous SCC cell line using retrovirl short hirpin RNA sequences enling stle Axl knock-down. We show tht, lthough Axl knock-down hs no effect on cell prolifertion, it sensitizes cells to -induced poptosis through incresed ctivtion of the pro-poptotic protein Bd, chnge in the conformtion of Bx nd Bk, relese of cytochrome c into the cytosol, nd ctivtion of cspses. These events re ccompnied y fster Akt dephosphoryltion in -treted Axl knockdown cells nd correlte with the degree of Axl knock-down. Tretment with the pn-cspse inhiitor zvadfmk prtilly rescued cells from -induced poptosis ut did not ffect Bid clevge or cytochrome c relese, suggesting tht cells die vi the mitochondril-medited pthwy. Thus, Axl confers resistnce of SCC cells to poptosis nd displys potentil s trget for therpeutic intervention in cutneous SCC. Journl of Investigtive Dermtology (211) 131, 59517; doi:1.138/jid ; pulished online 11 Novemer 21 INTRODUCTION Squmous cell crcinom (SCC) ccounts for pproximtely 2% of ll cutneous mlignncies nd occurs on exposed ody sites (Diepgen nd Mhler, 22). Aout 4% of cutneous SCCs metstsize (Brntsch et l., 28) nd these ptients hve 5-yer survivl of 25%, highlighting the need to improve therpy. We recently reported tht expression of the receptor tyrosine kinse, Axl, is upregulted in SCC skin tumors in vivo (Green et l., 26). Overexpression of Axl hs een ssocited with invsion nd metstsis in vrious cncers (Meric et l., 22; Singhi et l., 25; Ly et l., 27; Swu et l., 27). Axl, in conjunction with its lignd, growth rrest-specific gene 6 (Gs6), cn enhnce cell survivl. Recent dt hve shown tht Axl signling cn 1 Centre for Cutneous Reserch, Blizrd Institute of Cell nd Moleculr Science, Brts nd the London School of Medicine nd Dentistry, Queen Mry University of London, London, UK; 2 Deprtment of Nturl Sciences, Middlesex University, Enfield, UK nd 3 Deprtment of Moleculr Oncology, The Wetherll Institute of Moleculr Medicine, John Rdcliffe Hospitl, Oxford, UK Correspondence: Edel A. O Toole, Centre for Cutneous Reserch, Blizrd Institute of Cell nd Moleculr Science, Brts nd the London School of Medicine nd Dentistry, Queen Mry University of London, 4 Newrk Street, London E1 2AT, UK. E-mil: e..otoole@qmul.c.uk Received 9 Decemer 29; revised 18 August 21; ccepted 9 Septemer 21; pulished online 11 Novemer 21 prolong mlignnt gliom growth in mouse rin (Vjkoczy et l., 26). Following cell deth signl, numer of poptotic fctors, including cytochrome c, re relesed from the mitochondril intermemrne spce into the cytosol, y mechnism tht llows selective permeiliztion of the outer mitochondril memrne (OMM) (Kluck et l., 1997). The nti-poptotic proteins, Bcl-2, Bcl-X L, nd Mcl-1, ntgonize the relese of cytochrome c from the mitochondri nd susequent cspse ctivtion, wheres the propoptotic proteins, Bx nd Bk, promote poptosis (Wei et l., 21). Inhiitory molecules, such s Bd, ind to ntipoptotic Bcl-2/Bcl-X L proteins, therey preventing their sequestrtion of molecules such s cleved Bid (tbid) or Bim tht cn directly ctivte Bx nd Bk (Leti et l., 22; Kuwn nd Newmeyer, 23; Kim et l., 26). Immunohistochemicl nlysis reveled incresed phospho-akt (P-Akt) stining with significntly lower TUNEL stining in Axl-positive tumors nd led us to exmine the mechnisms y which Axl regultes poptosis in cutneous SCC cells. Retrovirl gene delivery of Axl-trgeting short hirpin RNA (shrna) sequences ws used to chieve stle Axl knock-down. Our results demonstrte tht Axl contriutes to the mlignnt SCC phenotype y protecting cells from -induced poptosis through modultion of the Akt nd ssocited PTEN pthwys nd suppression of the pro-poptotic ctivity of Bcl-2 fmily memers. & 211 The Society for Investigtive Dermtology 59

2 RESULTS Negtive correltion etween Axl protein expression nd poptosis in cutneous SCC tumors As Axl hs een reported to signl vi Akt in vrious cncers, therey protecting them from poptosis, we hypothesized tht there my e correltion etween Axl, P-Akt, nd poptosis in cutneous SCC tumors. Sections from 15 SCCs were scored for stining intensity etween (no stining) nd 3 (intense stining). In contrst to norml skin, which expresses miniml P-Akt, 11/15 (73%) SCCs exhiited P-Akt stining. Axl stining ws detected in 1/15 (67%) SCCs, wheres 6/15 (4%) SCCs showed poptotic cells detected y TUNEL stining. Stining for P-Akt ws strong in Axl-positive tumors nd ws ccompnied y significntly decresed TUNEL stining, wheres Axl-negtive tumors exhiited wek P-Akt nd strong TUNEL stining (Figure 1). Smples were sudivided into positive (23) nd wek or negtive (1) Axl stining for nlysis. Medin vlues (rs±sd) on the grph correspond to P-Akt or TUNEL stining in the Axl-positive nd -negtive tumors (Figure 1). There ws no correltion etween Axl expression nd differentition sttus. TUNEL P-Akt Axl Axl-positive tumor Axl-negtive tumor Knock-down of Axl sensitizes MET1 SCC cells to -induced poptosis In order to investigte the role of Axl in poptosis, sustined Axl knock-down ws chieved in MET1 SCC cells using shrna sequences 153 nd. Immunolotting of totl cell lystes from 153- nd -trnsduced cells reveled 49% nd 92% men Axl knock-down, respectively (Figure 2), without ny effect on cell prolifertion (Figure 2). Mitochondril poptosis involves ctivtion of cspse 3, which is responsile for proteolytic clevge of the nucler enzyme poly(adp-riose) polymerse (PARP) to ring out cell disssemly (Kufmnn et l., 1993). Immunolotting showed tht tretment with up to 24 hours resulted in smll fluctution of Axl expression levels (Figure 2c), which vried etween replicte experiments. Moreover, we oserved greter clevge of cspse 3 nd PARP in Axl knock-down compred with psuperior.retro.puro vector () cells (Figure 2c). Findings were replicted in second cell line, SCC-IC 1 (see Supplementry Figure S1 online). Apoptosis incresed over time, so tht 24 hours fter tretment there were 17%, 27% 153, nd 5% poptotic cells (Figure 2d). Thus, rogtion of Axl expression leds to incresed sensitivity to -induced poptosis, correlting with the degree of Axl knock-down. Knock-down of Axl increses sl phosphoryltion of PTEN nd the pro-poptotic potentil of Bd We next investigted the effect of Axl knock-down on Akt nd ssocited pthwys in SCC cells. Immunolotting showed pronounced time-dependent decrese in Akt phosphoryltion in response to tretment, which ws proportionl to the degree of Axl knock-down, wheres totl Akt levels remined unchnged (Figure 3). A downstrem Akt trget tht hs criticl role in mediting poptosis is the pro-poptotic protein Bd (Zh et l., 1996; Dtt et l., 1997). Totl levels of Bd expression remined lrgely Stining intensity unchnged y tretment, wheres there ws greter decline in the phosphoryltion of Bd in MET1 cells (Figure 3). Dephosphoryltion of PTEN, key tumor suppressor, t the C-terminl til, decreses its stility while enhncing its function s n ntgonist of PI3K/Akt signling (Stmolic et l., 1998; Vzquez et l., 2). Immunolotting reveled tht knock-down of Axl enhnced sl phosphoryltion of phospho-pten. In ddition, greter decrese in the totl expression levels of PTEN ws oserved over the time course of tretment in Axl knock-down cells (Figure 3). In order to verify the role of Akt in modulting resistnce to poptosis, MET1 cells were pre-treted with SH5, selective inhiitor of Akt ctivity (Figure 3). This Negtive (1) Positive (23) Axl stining intensity P-Akt TUNEL Figure 1. Correltion etween Axl, P-Akt, nd TUNEL stining in series of SCC tumors. () Stining for P-Akt nd TUNEL in Axl-positive nd -negtive SCC tumors. () Around 15 tumors were stined for Axl, P-Akt, nd TUNEL. The intensity of stining ws scored from (no stining) to 3 (intense stining). Smples were sudivided into positive (23) nd wek or negtive (1) Axl stining. Medin vlues (rs±sd) on the grph correspond to P-Akt or TUNEL stining in the positive nd negtive cells. Po.1. Br ¼ 1 mm. 51 Journl of Investigtive Dermtology (211), Volume 131

3 153 c 153 kd Axl 14 - Axl GAPDH 32 - Cpse 3 (N) Axl expression (% of control) * Cspse 3 (C) PARP (N) PARP (C) Tuulin Cell numer (fold increse) Dy d i Propidium iodide Untreted 6 Annexin V-FITC ii % Annexin-Vpositive cells Untreted 6 16 Time post- (h) * 24 Figure 2. Axl knock-down renders MET1 cells susceptile to poptosis. () Axl expression ws nlyzed y immunolot of cell lystes from MET1 cells trnsduced with vector lone or Axl-trgeting shrna sequences 153 or (irrelevnt lnes etween nd 153 were removed). () Cell prolifertion ws ssessed y counting cells using trypn lue exclusion t the indicted times. Dt shown re from three independent experiments. (c), 153, nd cells were exposed to (2 mj cm 2 ) nd expression levels of Axl, cspse 3, nd PARP were detected in cell lystes y immunolotting. Tuulin ws used s loding control. The dt re representtive of three independent experiments. (d) Apoptosis ws quntified y Annexin V-FITC/PI stining of, 153, nd cells treted with for the indicted times. (i) Sctter plots from one experiment re shown; (ii) dt (men±sem) from two experiments expressed s % Annexin-V-positive cells re shown. C, cleved; N, ntive. Po.1, *Po.1. resulted in significnt increse in poptosis 16 hours fter tretment with, which correlted with the degree of Axl knock-down, suggesting tht Axl functions vi Akt to protect SCC cells from poptosis (Figure 3c). Knock-down of Axl enhnced -induced ctivtion of Bx nd Bk in MET1 cells The pro-poptotic proteins Bx nd Bk regulte mitochondril cytochrome c relese through the formtion of pores in the OMM (Wei et l., 21), while Bcl-2, Bcl-X L, nd Mcl-1 re ntgonists of Bx nd Bk ctivtion (Kim et l., 26). Immunolotting showed no chnge in Bcl-2 or Bcl-X L expression nd Mcl-1 ws degrded eqully in response to tretment in oth Axl knock-down nd cells (Figure 4), indicting its dissocition from Bk, which is known to trigger poptosis (Nijhwn et l., 23). Although expression levels of Bk remined reltively unltered, there ws n increse in the expression of Bx over the time course of tretment (Figure 4). Activtion of Bx nd Bk ws exmined y conformtion-specific monoclonl ntiodies. Axl knock-down cells exhiited greter Bk nd Bx ctivities compred with cells, proportionl to the degree of Axl knock-down, in response to with mximl ctivtion t 16 hours (Figure 4 nd c). We next exmined the trnsloction of ctive monomeric Bx from the cytosol to the mitochondri y confocl microscopy. Results showed tht movement of Bx to the mitochondri (yellow stining) occurred within 6 hours of tretment nd ws greter in compred with cells (Figure 4d). These dt show tht knock-down of Axl in MET1 SCC cells results in chnge in the conformtion of Bk nd Bx in response to tretment. Incresed cytochrome c relese in the cytosol of -treted Axl knock-down cells To mesure mitochondril memrne potentil (Dcm), cells were stined with the potentil-sensitive dye tetrmethylrhodmine ethyl ester perchlorte (TMRE) nd 4,6-dimidino-2- phenylidole (DAPI) efore FACS processing; DAPI-positive cells were gted out for the nlysis. Tretments with NNO 3 or CCCP were used s positive controls. An lmost equl drop in Dcm of pproximtely 3% ws oserved s erly s 16 hours fter tretment in ll cells (Figure 5). To exmine cytochrome c relese into the cytosol, cells were permeilized with hypertonic digitonin-contining solution. Immunolotting exhiited greter cytochrome c relese in the cytosol of Axl knock-down compred with cells (Figure 5). These results show tht lthough knock-down of Axl does not seem to ffect Dcm, it results in cytochrome c relese into the cytosol in regulted wy

4 Axl knock-down enhnces cspse ctivtion nd leds to cspse-dependent poptosis To further determine the role of Axl in poptosis following cytochrome c relese, the ctivities of cspses 3 nd 8 were mesured in response to. Dt reveled tht cspse 3 ctivity in, 153, nd cells incresed y 4.-, 6.5-, nd 8.-fold, respectively, fter 24 hours of tretment (Figure 6). Similrly, cspse 8 ctivity incresed y chnge chnge chnge chnge chnge chnge DMSO % SH5 (µm) P-Akt Ser 473 Akt GAPDH P-Akt Ser 473 Akt P-Bd Ser 136 Bd P-PTEN Ser 38 PTEN Tuulin 2.5-, 4.-, nd 5.6-fold in, 153, nd, respectively (Figure 6). Activity of oth ws completely locked when cells were treted with for 24 hours in the presence of the pn-cspse inhiitor zvad-fmk (Figure 6 nd ). These results indicte tht -induced cspse ctivtion increses with the degree of Axl knock-down. Next, MET1 cells were treted with in the presence of zvad-fmk nd poptotic cells were quntified y Annexin V-FITC/PI stining. Our dt show tht -induced cell deth is prtly cspse dependent (Figure 6c). Knock-down of Axl ffects expression levels of Bid Bid is key pro-poptotic BH3-only molecule linking the receptor- nd mitochondri-medited poptotic pthwys. Clevge nd ctivtion of ntive p22 Bid y cspse 8 results in the genertion of p15 tbid, which hs role in the ctivtion of Bx nd Bk nd cytochrome c relese (Luo et l., 1998). Immunolotting of totl cell lystes reveled striking decrese in the sl expression levels of Bid, which ws proportionte to the degree of Axl inhiition in MET1 cells (Figure 6d). However, levels of tbid were similr in Axl knock-down nd cells 24 hours fter tretment with (Figure 6d). Thus, lthough Axl seems to hve role in modulting Bid expression, it does not seem to ffect Bid processing. As n increse in cspse 8 ctivity ws oserved in response to tretment in MET1 cells (Figure 6), we wnted to clrify the role of Bid in -induced deth in SCC cells. Immunolot nlysis reveled tht lthough tretment with zvad-fmk inhiited proteolytic processing of Bid, it did not ffect the mount of cytochrome c relesed in response to (Figure 6e). Inhiition of Bid expression using trnsient smll interfering RNA (sirna) trnsfection (Figure 6f) exhiited no significnt chnge in -induced poptosis (Figure 6f). These dt indicte tht cytochrome c relese occurs upstrem of cspse ctivtion, wheres proteolytic processing of Bid ppers to e downstrem event. DISCUSSION A mjor feture of SCC cells is their resistnce to poptosis (Er et l., 25). Immunohistochemicl nlysis of SCC tumors reveled correltion etween Axl nd P-Akt stining with reduction in TUNEL stining in Axl-positive tumors, leding us to investigte further the mechnism y which Axl promotes tumor cell survivl. Knock-down of Axl in MET1 c Cspse 3 ctivity per cell titer (fold increse) SH5 153 Figure 3. Effect of Axl knock-down on the ctivtion of Akt, Bd, nd PTEN in response to. (), 153, nd MET1 cells were exposed to (2 mj cm 2 ) for the indicted times nd expression levels of Akt, P-Akt Ser 473, Bd, P-Bd Ser 136, PTEN, nd P-PTEN Ser 38 were detected in totl cell lystes y immunolotting. Tuulin ws used s loding control. Br grphs show the fold chnge (men±sd) in protein expression from two to three lots. () Immunolot nlysis showing expression levels of Akt nd P-Akt Ser 473 fter doseresponse tretment using the specific inhiitor of Akt ctivity SH5. DMSO ws used s vehicle control, while GAPDH ws used s loding control. (c), 153, nd MET1 cells were pre-treted with SH5 (4 mm) for 1 hour nd were susequently treted with (2 mj cm 2 ) for 16 hours. Apoptosis ws determined y clculting the rtio of cspse 3 ctivity over cellulr ATP content (cell titer). Dt (men±sem) re expressed s fold chnge of respective controls. Po Journl of Investigtive Dermtology (211), Volume 131

5 153 i Bk Bx Counts c Bcl-X L Bcl-2 Mcl-1 GAPDH PE 575/26nm-A ii Fluorescence intensity (fold of control) d Untreted * Time post- (h) 6 16 IP:Bx (6A7) IB:Bx (P-19) IP:input Bx (P-19) Tuulin IgG (light chin) Figure 4. tretment results in incresed ctivtion of Bx nd Bk in Axl knock-down MET1 cells. () MET1, 153, nd cells were exposed to (2 mj cm 2 ) nd expression levels of Bk, Bx, Bcl-X L, Bcl-2, nd Mcl-1 were detected in totl cell lystes y immunolotting. () -induced ctivtion of Bk ws detected y hrvesting nd immunostining MET1 cells with the conformtion-specific nti-bk A-1 ntiody followed y FACS nlysis. (i) Histogrms from one experiment re shown; (ii) dt (men±sem) re expressed s fold of respective controls from two independent experiments. *Po.5. (c) Active monomeric Bx ws detected y immunoprecipittion using the monoclonl conformtion-specific nti-bx 6A7 ntiody followed y immunolotting with the polyclonl nti-bx P-19 ntiody. (d) Confocl microscopy ws performed using the nti-bx 6A7 ntiody nd the mitochondril dye mitotrcker ornge. Bx (green) locliztion to the mitochondri (red) in response to is shown in yellow. Nuclei were counterstined with DAPI (lue). A representtive imge of the stining pttern is shown from three seprte experiments. Br ¼ 1 mm. SCC cells resulted in significntly greter cspse ctivity nd poptosis in response to compred with control cells. A previous study using hed nd neck SCC cell line demonstrted tht one of the most significnt repercussions of deregulted Akt signling is disruption of the poptotic response to B (Decrene et l., 24). Knock-down of Axl did not mjorly ffect totl or phosphorylted sl levels of Akt. However, Axl knock-down resulted in pronounced loss of -induced Akt phosphoryltion nd ws ccompnied y dephosphoryltion of the BH3-only protein Bd, which is known to enhnce the ility of Bd to ntgonize Bcl-2 nd Bcl-X L nti-poptotic ctivities (Zh et l., 1996; Dtt et l., 1997). Our findings re consistent with previous studies showing tht Axl functions vi Akt to enhnce cell survivl (Goruppi et l., 1999; Lee et l., 22; Swu et l., 27). To further clrify the mechnism of Akt dephosphorltion, we focused on PTEN, known ntgonist of Akt-dependent cell survivl (Stmolic et l., 1998). Axl hs previously een shown to interct with C1-TEN, PTEN homolog (Hfizi et l., 22); however, no ssocition hs 513

6 Δψm (% of mx) Untreted CCCP NNO Cyt. c (cytosolic) Tuulin (cytosolic) Cyt c (totl) Tuulin (totl) Figure 5. Incresed -induced cytochrome c relese in Axl knock-down cells. () Cells were hrvested fter 16 hours of tretment (2 mj cm 2 ). Tretment with cronyl cynide 3-chlorophenylhydrzone (CCCP) or NNO 3 for 2 hours ws used s positive control for mitochondril depolriztion. A chnge in mitochondril memrne potentil (Dcm) ws ssessed y tetrmethylrhodmine ethyl ester perchlorte nd DAPI stining followed y FACS nlysis. Dt (men±sem) re expressed s percent of mximum of respective controls. () Cytochrome c ws detected in digitoninextrcted cytosolic frctions nd in totl cell lystes y immunolotting. Dt re representtive of three independent experiments. een reported etween Axl nd PTEN. Interestingly, our results reveled tht knock-down of Axl mrkedly incresed sl PTEN phosphoryltion nd led to decrese in totl PTEN levels in response to tretment. Recent studies hve reported tht the ctivity of PTEN is relted to its phosphoryltion sttus t the C-terminl til, with dephosphoryltion resulting in decresed protein stility with coincident increse in ctivity (Vzquez et l., 2, 21). The incresed phosphoryltion of PTEN in the Axl knockdown cells could explin the loss of Akt phosphoryltion in response to tretment, positioning Axl s negtive regultor of PTEN ctivity. We next investigted the effect of knocking down Axl on the ctivities of Bx nd Bk, two proteins tht re required for commitment to cell deth (Wei et l., 21). A chnge in the conformtion of Bx nd Bk ws detected within 6 hours of tretment. This ws ccompnied y cytochrome c relese, which ws greter in Axl knock-down cells. This would imply tht cytochrome c relese tkes plce in regulted mnner through the formtion of specific chnnels involving Bx nd Bk nd is negtively regulted y Axl. Exmintion of nti-poptotic proteins reveled tht consistent with previous studies (Nijhwn et l., 23), Mcl-1 ws eqully down-regulted in response to in nd Axl knock-down cells. Contrry to previous report (Qin et l., 26), tretment did not lter expression levels of Bcl-2 nd Bcl-X L in MET1 cells. In n ttempt to chrcterize further the mechnism y which Axl modultes poptosis, the expression of Bid, crucil protein in the crosstlk etween receptor- nd mitochondri-medited pthwys, ws exmined. Bsl expression of Bid ws reduced in Axl knock-down cells, suggesting tht Axl my hve role in regulting Bid stility. Bid clevge ecme evident fter cytochrome c relese hd een detected nd ws proportionl to the degree of Axl knock-down. Additionlly, inhiition of cspses locked clevge of Bid, ut did not ffect cytochrome c relese into the cytosol, indicting it is unlikely for this process to hve een driven y Bid clevge. Thus, clevge of Bid likely occurs downstrem of Bx nd Bk ctivtion nd is ctlyzed y cspses. Contrry to previous studies, which hve descried Bid clevge s n mplifiction step to the poptotic process downstrem of cytochrome c relese (Slee et l., 2), our dt using sirna to trget Bid expression revel redundnt role for Bid in induced cell deth in ccordnce with previous studies (Kufmnn et l., 27). Our dt suggest tht SCC cells die predominntly vi the mitochondri-medited pthwy. In summry, our dt demonstrte tht Axl controls the reltive lnce etween Akt nd PTEN ctivities to modulte the poptotic response in cutneous SCC through negtive regultion of Bk nd Bk nd to lesser extent the BH3-only protein Bd, providing mechnism for the role of Axl in cutneous SCC resistnce to poptosis. Becuse Axl is locted t the cell memrne, successful inhiition of its ctivity will potentilly decrese downstrem intrcellulr signling nd impede tumor development without the risk ssocited with intrcellulr feedck loops. The vlue of Axl s therpeutic trget hs een vlidted using potent Axl kinse inhiitors tht inhiited rest cncer cell motility nd invsion (Zhng et l., 28). More recently, R428, selective smll-molecule inhiitor of Axl, hs een demonstrted to prolong survivl in niml models of metsttic rest cncer (Hollnd et l., 21). Our dt support role for Axl s trget for therpeutic intervention in cutneous SCC. MATERIALS AND METHODS Cell culture nd tretments Spontneously immortlized SCC cells (MET1 nd SCC-IC1) (Proy et l., 2; Mrtins et l., 29) were cultured s descried previously (Rheinwld, 198). The pn-cspse inhiitor zvad-fmk (Promeg, Southmpton, Hmpshire, UK) or the Akt inhiitor SH5 (Enzo Life Sciences, Exeter, UK) ws dded to the medi 1 hour efore stimultion nd during tretment. 514 Journl of Investigtive Dermtology (211), Volume 131

7 Cspse 3 ctivity (fluorescence units) 1,5 1, * * * Cspse 8 ctivity (fluorescence units) * * * * e % Annexin-V-positive cells Control zvad-fmk c Time post- (h) * zvad-fmk Negtive d Time post- (h) f Control Bid sirna NT sirna Bid Tuulin Bid tbid GAPDH Negtive Cyt c (cytosolic) Tuulin (cytosolic) Cyt c (totl) Bid (totl) Tuulin (totl) g Cspse 3 ctivity per cell titer (fold increse) NT sirna Bid sirna 153 NT sirna 153 Bid sirna NT sirna Bid sirna 16 Time post- (h) Figure 6. Axl knock-down diminishes expression of Bid, protein tht hs redundnt role in cspse-dependent cell deth. MET1, 153, nd cells were treted with for the indicted times in the presence or sence of zvad-fmk (25 mm), nd ctivities of () cspse 3 nd () cspse 8 were quntified y incuting totl cell lystes with specific fluorogenic sustrtes. (c) Apoptosis ws quntified y Annexin V-FITC/PI stining of, 153, nd cells treted or not with (2 mj cm 2 ) nd/or zvad-fmk for 16 hours. Dt (men±sem) shown re from three independent experiments. *Po.5, Po.1. (d) Cells were hrvested fter tretment nd expression levels of full-length Bid nd tbid were detected in totl cell lystes y immunolotting. (e) Cells were pre-incuted with zvad-fmk nd were susequently treted with for the indicted times. Cytosolic nd totl cytochrome c nd totl Bid levels were exmined y immunolotting. (f), 153, nd MET1 cells were trnsiently trnsfected with Bid or non-trgeting (NT) sirna (5 nm) nd immunolot nlysis for Bid ws crried out 96 hours post-trnsfection. Tuulin ws used s loding control. (g) Bid sirna -ornt sirna -trnsfected MET1 cells were treted with for 16 hours nd poptosis ws determined y clculting the rtio of cspse 3 ctivity over cellulr ATP content (cell titer). Dt (men±sem) re expressed s fold chnge of respective controls

8 RNA interference, retrovirl genertion nd delivery Oligonucleotides corresponding to different trget sequences were generted using OligoEngine RNAi Design Tool (Amion, Huntingdon, UK). shrna trget sequences re listed elow with their strting positions in the coding region of Axl: (153) 5 -GACATCCTC TTTCTCCTGC-3 nd () 5 -GATTTGGAGAACACACTGA-3. Anneled oligonucleotides were cloned into linerized y BglII nd HindIII downstrem of the H1 promoter TATA ox. or shrna-contining plsmids were trnsfected into Phoenix pckging cells (Origen, Sn Diego, CA) using FuGene 6 (Roche, Welwyn Grden City, UK). At 2 dys post trnsfection, cells were incuted in DMEM supplemented with 1% fetl ovine serum, 2mML-glutmine, nd 1.25 mgml 1 puromycin. Puromycin-resistnt cells were mintined in selection medi nd were sucultured when 8% confluent. Confluent cells were plced in humidified incutor t 32 1C for 12 hours to produce retrovirus-contining superntnt, which ws used to infect SCC cells s descried (Akgul et l., 25). Trnsient cell trnsfections of non-trgeting or Bid ON-TARGETplus SMARTpool sirna (5 nm) sequences were performed using Dhrmfect s descried previously (Mrtins et l., 29). B irrdition The B source ws P Multiple Ry Lmp (Ultr-violet Products, Cmridge, UK) (MRL-58 model) with emission t 32 nm, which ws clirted efore ech experiment. Cells were then replenished with fresh medi. Cell extrcts nd immunolotting Cells were lysed in Triton lysis uffer (TLB) s descried previously (Tournier et l., 2). Cytosolic extrcts were prepred using mnnitol uffer with digitonin (8 mgml 1 ) s descried (Lee et l., 26) nd were concentrted using Microcon Bioseprtions centrifugl filters (Millipore, Wtford, UK). Protein content ws normlized using Bio-Rd DC ssy. Following SDS-PAGE, proteins were trnsferred onto Hyond-P memrne (Bio-Rd Lortories, Hemel Hempsted, UK) using wet trnsfer. Memrnes were sturted in Tris-uffered sline, ph 7.6, with.1% Tween 2 (Sigm-Aldrich Compny, Dorset, UK) nd 5% non-ftdried milk (Mrvel, Premier Foods, St Alns, UK). Antiodies ginst Akt, P-Akt Ser 473,AIF,Bd,cspse3,PARP,Bcl-2,ndMcl-1fromCell Signlling Technology (Dnvers, MA), Bcl-X L (H-62),Bx(P-19),Axl (C-2), phospho-pten (P-PTEN) Ser 38 nd P-Bd Ser 136 from Snt Cruz Biotechnology (Snt Cruz, CA), PTEN from Millipore, Bid from R&D Systems (Minnepolis, MN), cytochrome c from BD Biosciences (Oxford, UK), Bk (A-1), tuulin from EMD Chemicls (Gistown, NJ) nd GAPDH from Acm (Cmridge, UK) were used for immunodetection. Secondry ntiodies coupled to horserdish peroxidse were from Dko. Signls were detected y ECL þ (GE Helthcre Life Sciences, Buckinghmshire, UK). Densitometry ws performed y ImgeJ 1.41 nd vlues were normlized to totl protein nd expressed s fold chnge. FACS nlysis Apoptosis ws detected using TACS Annexin V-FITC (R&D Systems). Mitochondril memrne depolriztion ws ssessed y uptke of TMRE (Invitrogen, Pisley, UK). Cronyl cynide 3-chlorophenylhydrzone (CCCP, 1 mm) nd NNO 3 (1% v/v) were used s positive controls. TMRE (4 nm) ws dded to the medi 3 minutes efore the end of tretment nd cells were trypsinized nd wshed with PBS. Cell pellets were resuspended in HEPES (25 mm) nd DAPI (5 ng ml 1 )ws dded 1 minutes efore FACS nlysis. To ssess ctivtion of Bk, cells were fixed with PBS/.25% prformldehyde, hrvested y scrping nd permeilized with PBS/.1% sponin. Cells were incuted with nti- Bk A-1 mouse monoclonl ntiody (1/5 dilution) nd then phycoerythrin-conjugted (Dko, 5 mg l 1 ; 1/3 dilution) ntiody while mixing (4 1C, 3 minutes). Mouse IgG ws used s negtive control. Anlysis ws performed with Becton Dickinson LSRII flow cytometer (BD Biosciences) using BD FACSDiv 6. softwre. Cspse, viility, nd prolifertion ssys Activities of cspses 3 nd 8 were mesured fluorometriclly using Ac-DMQD-AMC or Ac-LETD-AFC sustrtes (Enzo Life Sciences) following the mnufcturer s instructions. Sustrte clevge ws ssessed y monitoring fluorescence intensity. Cell viility nd cspse 3 ctivity of cells grown in 96-well pltes (3, cells per well) were mesured using CellTitre-Glo nd Cspse-Glo 3/7 Luminescent ssys (Promeg), respectively, ccording to the mnufcturer s instructions. The intensity of fluorescence nd luminescence ws mesured using Synergy 4 Multi-Mode Microplte Reder. The rtio of cspse 3/7 ctivity over cellulr ATP content (cell titer) ws used s mesure of poptosis. Cell prolifertion ws determined y trypsinizing nd counting cells exhiiting trypn lue dye exclusion. Immunoprecipittion Protein A grose Fst Flow eds (Upstte) were mixed with 2 mg nti-bx 6A7 ntiody (Sigm) on rotting wheel (4 1C, 2 hours). Antiodyed complexes were wshed thrice in TLB, comined with pre-clered cell lystes, nd immunoprecipitted on rotting wheel (4 1C, overnight). Antigenntiody complexes were wshed four times in TLB nd the rection ws stopped y dding 6 Lemmli uffer nd heting the smples (95 1C, 5 minutes). Histology nd immunostining Archivl prffin locks were used with ethics pprovl otined from the Est London nd City Helth Authority Reserch Ethics Committee. Expression of Axl nd P-Akt Ser 473 ws exmined using immunohistochemistry on 4 mm deprffinized sections locked with horse serum. Immunostining ws performed s descried previously (Jckson et l., 22). Apoptotic cells were detected in SCC sections y TUNEL ssy (Ded-End, Promeg). Quntittive nlysis of stining ws performed using KS4 version 3. imging softwre (Crl Zeiss, Welwyn Grden City, UK). Stining intensity ws scored y counting cells in four representtive high-power fields, nd ws grded s (no stining), 1 (wek), 2 (moderte), nd 3 (intense). The nti-bx 6A7 ntiody ws used to detect ctive monomeric Bx in cells. Mitochondri were stined with 1 nm mitotrcker ornge (Moleculr Proes) for 3 minutes in humidified tmosphere of 37 1C, 1% CO 2, nd 9% ir. Acetone: methnol (1:1)-fixed cells were locked in PBS,.1% Triton X-1, 3% (w/v) ovine serum lumin, nd incuted with primry (4 1C, overnight) nd AlexFluor 488-conjugted (Moleculr Proes) secondry ntiodies (room temperture, 1 hour). Nuclei were counterstined with DAPI (1 mgml 1 ) nd smples were mounted onto glss slides using Immu-Mount (Thermo Fisher Scientific, Wlthm, MA). Imges were cptured using Zeiss LSM 51 META lser-scnning microscope. Sttisticl nlysis Sttisticl significnce ws determined y one-wy nlysis of vrince (ANOVA), with post hoc Tukey s test for comprison 516 Journl of Investigtive Dermtology (211), Volume 131

9 etween groups. Two-wy ANOVA with Bonferroni correction for comprison etween replicte mens ws used for time-course tretments. CONFLICT OF INTEREST The uthors stte no conflict of interest. ACKNOWLEDGMENTS This work ws funded y the Assocition for Interntionl Cncer Reserch (3-315), the British Skin Foundtion, nd Der Irelnd. We would like to cknowledge the generous gift of SCC cell lines from Irene Leigh nd Chrlotte Proy, Cncer Reserch UK Skin Tumour Lortory, nd Ver Mrtins for help with photogrphy. SUPPLEMENTARY MATERIAL Supplementry mteril is linked to the online version of the pper t REFERENCES Akgul B, Grci-Escudero R, Ghli L et l. (25) The E7 protein of cutneous humn ppillomvirus type 8 cuses invsion of humn kertinocytes into the dermis in orgnotypic cultures of skin. Cncer Res 65: Brntsch KD, Meisner C, Schonfisch B et l. (28) Anlysis of risk fctors determining prognosis of cutneous squmous-cell crcinom: prospective study. Lncet Oncol 9:7132 Dtt SR, Dudek H, To X et l. (1997) Akt phosphoryltion of BAD couples survivl signls to the cell-intrinsic deth mchinery. Cell 91:23141 Decrene D, Vn Lethem A, Agostinis P et l. (24) AKT sttus controls susceptiility of mlignnt kertinocytes to the erly-ctivted nd Binduced poptotic pthwy. J Invest Dermtol 123:2712 Diepgen TL, Mhler V (22) The epidemiology of skin cncer. Br J Dermtol 146(Suppl 61):16 Er P, Ji J, Wernli M et l. (25) Role of poptosis in sl cell nd squmous cell crcinom formtion. Immunol Lett 1:6872 Goruppi S, Ruro E, Vrnum B et l. (1999) Gs6-medited survivl in NIH3T3 cells ctivtes stress signlling cscde nd is independent of Rs. Oncogene 18: Green J, Ikrm M, Vys J et l. (26) Overexpression of the Axl tyrosine kinse receptor in cutneous SCC-derived cell lines nd tumours. Br J Cncer 94: Hfizi S, Alindri F, Krlsson R et l. (22) Interction of Axl receptor tyrosine kinse with C1-TEN, novel C1 domin-contining protein with homology to tensin. Biochem Biophys Res Commun 299:7938 Hollnd SJ, Pn A, Frnci C et l. (21) R428, selective smll molecule inhiitor of Axl kinse, locks tumor spred nd prolongs survivl in models of metsttic rest cncer. Cncer Res 7: Jckson S, Ghli L, Hrwood C et l. (22) Reduced poptotic levels in squmous ut not sl cell crcinoms correltes with detection of cutneous humn ppillomvirus. Br J Cncer 87:31923 Kufmnn SH, Desnoyers S, Ottvino Y et l. (1993) Specific proteolytic clevge of poly(adp-riose) polymerse: n erly mrker of chemotherpy-induced poptosis. Cncer Res 53: Kufmnn T, Ti L, Ekert PG et l. (27) The BH3-only protein id is dispensle for DNA dmge- nd replictive stress-induced poptosis or cell-cycle rrest. Cell 129:42333 Kim H, Rfiuddin-Shh M, Tu HC et l. (26) Hierrchicl regultion of mitochondrion-dependent poptosis y BCL-2 sufmilies. Nt Cell Biol 8: Kluck RM, Bossy-Wetzel E, Green DR et l. (1997) The relese of cytochrome c from mitochondri: primry site for Bcl-2 regultion of poptosis. Science 275:11326 Kuwn T, Newmeyer DD (23) Bcl-2-fmily proteins nd the role of mitochondri in poptosis. Curr Opin Cell Biol 15:6919 Ly JD, Hong CC, Hung JS et l. (27) Sulfslzine suppresses drug resistnce nd invsiveness of lung denocrcinom cells expressing AXL. Cncer Res 67: Lee WK, Aouhmed M, Thevenod F (26) Cspse-dependent nd -independent pthwys for cdmium-induced poptosis in cultured kidney proximl tuule cells. Am J Physiol Renl Physiol 291:F82332 Lee WP, Wen Y, Vrnum B et l. (22) Akt is required for Axl-Gs6 signling to protect cells from E1A-medited poptosis. Oncogene 21:32936 Leti A, Bssik MC, Wlensky LD et l. (22) Distinct BH3 domins either sensitize or ctivte mitochondril poptosis, serving s prototype cncer therpeutics. Cncer Cell 2:18392 Luo X, Budihrdjo I, Zou H et l. (1998) Bid, Bcl2 intercting protein, medites cytochrome c relese from mitochondri in response to ctivtion of cell surfce deth receptors. Cell 94:4819 Mrtins VL, Vys JJ, Chen M et l. (29) Incresed invsive ehviour in cutneous squmous cell crcinom with loss of sement-memrne type VII collgen. J Cell Sci 122: Meric F, Lee WP, Shin A et l. (22) Expression profile of tyrosine kinses in rest cncer. Clin Cncer Res 8:3617 Nijhwn D, Fng M, Trer E et l. (23) Elimintion of Mcl-1 is required for the initition of poptosis following ultrviolet irrdition. Genes Dev 17: Proy CM, Purdie KJ, Sexton CJ et l. (2) Spontneous kertinocyte cell lines representing erly nd dvnced stges of mlignnt trnsformtion of the epidermis. Exp Dermtol 9:1417 Qin JZ, Xin H, Sitilo LA et l. (26) Enhnced killing of melnom cells y simultneously trgeting Mcl-1 nd NOXA. Cncer Res 66: Rheinwld JG (198) Seril cultivtion of norml humn epiderml kertinocytes. Methods Cell Biol 21A:22954 Singhi PP, Cstello L, Bergmsco L et l. (25) Gs6 induces prolifertion in prostte crcinom cell lines expressing the Axl receptor. J Cell Physiol 24:3644 Swu T, Seno H, Kwshim T et l. (27) Growth rrest-specific gene 6 nd Axl signling enhnces gstric cncer cell survivl vi Akt pthwy. Mol Crcinog 46:15564 Slee EA, Keogh SA, Mrtin SJ (2) Clevge of BID during cytotoxic drug nd rdition-induced poptosis occurs downstrem of the point of Bcl-2 ction nd is ctlysed y cspse-3: potentil feedck loop for mplifiction of poptosis-ssocited mitochondril cytochrome c relese. Cell Deth Differ 7:55665 Stmolic V, Suzuki A, de l Pomp JL et l. (1998) Negtive regultion of PKB/Akt-dependent cell survivl y the tumor suppressor PTEN. Cell 95:2939 Tournier C, Hess P, Yng DD et l. (2) Requirement of JNK for stressinduced ctivtion of the cytochrome c-medited deth pthwy. Science 288:874 Vjkoczy P, Knyzev P, Kunkel A et l. (26) Dominnt-negtive inhiition of the Axl receptor tyrosine kinse suppresses rin tumor cell growth nd invsion nd prolongs survivl. Proc Ntl Acd Sci USA 13: Vzquez F, Grossmn SR, Tkhshi Y et l. (21) Phosphoryltion of the PTEN til cts s n inhiitory switch y preventing its recruitment into protein complex. J Biol Chem 276: Vzquez F, Rmswmy S, Nkmur N et l. (2) Phosphoryltion of the PTEN til regultes protein stility nd function. Mol Cell Biol 2:518 Wei MC, Zong WX, Cheng EH et l. (21) Propoptotic BAX nd BAK: requisite gtewy to mitochondril dysfunction nd deth. Science 292:7273 Zh J, Hrd H, Yng E et l. (1996) Serine phosphoryltion of deth gonist BAD in response to survivl fctor results in inding to not BCL-X(L). Cell 87:61928 Zhng YX, Knyzev PG, Cheurkin YV et l. (28) AXL is potentil trget for therpeutic intervention in rest cncer progression. Cncer Res 68: