CD95L Cell Surface Cleavage Triggers a Prometastatic ... · Molecular and Cellular Pathobiology CD95L Cell Surface Cleavage Triggers a Prometastatic Signaling Pathway in Triple-Negative

Molecular and Cellular Pathobiology

CD95L Cell Surface Cleavage Triggers a PrometastaticSignaling Pathway in Triple-Negative Breast Cancer

Marine Malleter1,2, S�ebastien Tauzin1,2, Alban Bessede4, R�emy Castellano5, Armelle Goubard5,Florence Godey1,3, Jean Leveque1,2, Pascal J�ez�equel6, Loic Campion6, Mario Campone6, Thomas Ducret7,8,Ga€etan MacGrogan9, Laure Debure1,2, Yves Collette5, Pierre Vacher7,9, and Patrick Legembre1,2

AbstractTriple-negative breast cancers (TNBC) lacking estrogen and progesterone receptors and HER2 amplification

have a relatively high risk of metastatic dissemination, but the mechanistic basis for this risk is not understood.Here, we report that serum levels of CD95 ligand (CD95L) are higher in patients with TNBC than in other patientswith breast cancer. Metalloprotease-mediated cleavage of CD95L expressed by endothelial cells surroundingtumors generates a gradient that promotes cell motility due to the formation of an unconventional CD95-containing receptosome called the motility-inducing signaling complex. The formation of this complex wasinstrumental for Nox3-driven reactive oxygen species generation. Mechanistic investigations revealed a Yes–Orai1–EGFR–PI3K pathway that triggered migration of TNBC cells exposed to CD95L. Our findings establish aprometastatic function for metalloprotease-cleaved CD95L in TNBCs, revisiting its role in carcinogenesis. CancerRes; 73(22); 6711–21. �2013 AACR.

IntroductionHuman breast tumors are heterogeneous, both in their

clinical expression and molecular profiles. Triple-negativebreast cancers (TNBC) are characterized by negative immu-nohistochemical staining for estrogen and progesterone recep-tors and HER2, and represent 10% to 20% of all breast cancers.TNBCs account for early deaths of a disproportionate numberof patient due to their aggressive nature and the lack ofeffective therapeutic treatment options (1). Gaining insightsinto the molecular mechanisms that promote TNBC invasive-ness could be of significant value for the design of treatmentstrategies aimed at preventing metastatic dissemination ofthese cancers. In addition, prognostic markers are needed todiscriminate among those patients with TNBC who are athighest risk for metastatic relapse.CD95 ligand (CD95L, also known as FasL) belongs to the

TNF family and is the ligand for the "death receptor" CD95

(Fas/APO1). CD95L is a transmembrane "cytokine" whoseextracellular domain can be cleaved by metalloproteases (2),to produce a soluble ligand. This soluble form was initiallydescribed as an inert ligand that competes with its membrane-bound counterpart for binding to CD95, thus acting as anantagonist of the death signal (3, 4). More recent findings haveshown that metalloprotease-cleaved CD95L (cl-CD95L) canactively participate in aggravating inflammation in chronicinflammatory disorders, such as systemic lupus erythematosus(5, 6), andmay exert pro-oncogenic functions by promoting thesurvival of ovarian and liver cancers (7) and chemotherapyresistance of lung cancers (8) through molecular mechanismsthat remain to be elucidated.

Binding of transmembrane CD95L to CD95 leads to therecruitment of the adaptor protein Fas-associated deathdomain (FADD) to the intracellular region of CD95 called thedeath domain. In turn, FADD binds to caspases-8 and -10. ThisCD95/FADD/caspase complex is known as the death-inducingsignaling complex (DISC; ref. 9) and plays a pivotal role in theinitiation of the apoptotic signal. In contrast, cl-CD95L fails toinduce DISC formation and instead promotes the formation ofan atypical receptosome that we have designated motility-inducing signaling complex (MISC; ref. 6). MISC formationleads to the induction of the pro-oncogenic phosphoinositide3-kinase (PI3K) signaling pathway (6, 10) through a molecularmechanism that remains to be elucidated.

Here, we show that the level of cl-CD95L is higher in theblood of patients with TNBC than in that of patients withoutTNBC and is associated with increased risk of developingdistant metastases. Moreover, we demonstrate that aftercleavage by a metalloprotease, the soluble CD95L promotesthe motility of TNBC cells by inducing Nox3 (nicotinamideadenine dinucleotide phosphate-oxidase oxidase-3)-driven

Authors' Affiliations: 1Inserm U1085-IRSET, Equipe Labellis�ee LigueContre Le Cancer; 2Universit�e de Rennes-1; 3Centre Eug�ene Marquis, ruebataille Flandres Dunkerque, Rennes; 4ImmuSmol, 15 Rue Amiral Prouhet,Pessac; 5Inserm, U1068; Institut Paoli-Calmettes; Aix-Marseille Universit�e,CNRS,UMR7258,Marseille; 6ICO-Ren�eGauducheau, Bd J.Monod, Saint-Herblain; 7Universit�e de Bordeaux; 8Inserm U1045, Universit�e BordeauxSegalen; and 9Inserm U916, Institut Bergoni�e, Bordeaux, France

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

M. Malleter and S. Tauzin share first authorship.

Corresponding Author: Patrick Legembre, Universit�e de Rennes-1, 2Avenue Prof. L�eon Bernard, Rennes 35043, France. Phone: 33-2232-34807; Fax: 33-2232-34794; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-13-1794

�2013 American Association for Cancer Research.

CancerResearch

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reactive oxygen species (ROS) generation, which activates Srckinase c-yes, leading to PI3K signaling through activation ofthe EGF receptor (EGFR) in an EGF-independent manner.

Materials and MethodsEthics statement

All clinical investigation was conducted in accordance withthe principles outlined in the Declaration of Helsinki.

Cell lines, antibodies, plasmids, and other reagentsHuman breast adenocarcinoma cells MDA-MB-231, MDA-

MB-468, and Hs578T were maintained in Dulbecco' ModifiedEagle Medium supplemented with 8% (v/v) heat-inactivatedfetal calf serum and 2mmol/L L-glutamine at 37�C in a 5% CO2

incubator. Silencing experiments were carried out by lentiviraltransduction of TNBC cells using shRNAmir-pGIPZ vectors(Open Biosystems). All reagents are described in Supplemen-tary Materials and Methods.

Mouse experimentsNOD/SCID (nonobese diabetic/severe combined immuno-

deficient)/gc null mice (NSG) were obtained from CharlesRiver Laboratory (Margate, UK). Luciferase-expressing MDA-MB-231 cells resuspended with or without cl-CD95L (100ng/mL; 5� 105 cells in 50 mL PBS/Matrigel) were transplantedinto mammary fat pads of mice (7-week-old female). Next,caudal vein injections of cl-CD95L (10 mg/kg) or controlmedium were repeated 5 days a week until day 34. Biolumi-nescence analysis was performed using PhotonIMAGER (Bio-space Lab), following intraperitoneal injection of luciferin(30 mg/kg). Tumor volume was calculated using the formulaV ¼ 0:52 ðL�W2Þ. After completion of the analysis, autopsyof mice was done and organ luminescence was assessed.

DISC/MISC analysisTNBC cells (3 � 107 cells) were incubated with 100 ng/mL

of cl-CD95L (MISC) or APO1-3 (DISC) for the indicated times.The cells were lysed, 1 mg of Apo1-3 was added to the celllysate, and CD95 was immunoprecipitated by the addition ofprotein A-sepharose beads (Sigma). For the immunoprecipi-tation of EGFR, 1 mg of anti-EGFR (Santa Cruz Biotechnology)was added to the cell lysate and EGFR was immunoprecip-itated with protein A-sepharose beads. After extensive wash-ing, the immune complex was resolved by SDS-PAGE.

Ca2þ monitoringCa2þ monitoring was performed as previously described

(11).Statistical analyses and Supplementary Materials andMeth-

ods are available online.

ResultsSerum CD95L level predicts metastasis in breast cancers

Although high CD95L levels have been detected by immu-nohistochemistry in breast cancer tissue (12), the role of thisligand in carcinogenesis remains unknown. To address thisquestion, serum CD95L levels were measured in women withbreast cancer. Blood dosages showed higher CD95L levels in

women affected byTNBC than in patientswithout TNBC (98.94� 45.37,n¼ 39 vs. 52.79� 26.2, n¼ 103;P< 0.0001) and subjectswith benign breast diseases (98.94 � 45.37, n ¼ 39 vs. 30.04 �28.52, n¼ 8; P < 0.0001; Fig. 1A). Of note, the quantity of CD95Lexhibited a heterogeneous distribution in patients withoutTNBC (Fig. 1A); however, when this cohort of patients wassubdivided on the basis of the occurrence of relapse, CD95Llevels were significantly higher in relapsing patients than inthose without relapse (62.72� 31.31, n¼ 39 vs. 47.68� 21.77, n¼ 64; P ¼ 0.0053; Fig. 1A). In addition, Kaplan–Meier analysesrevealed that both patients with and without TNBC withCD95L concentrations �80 pg/mL had significantly reduceddisease-free survival (Fig. 1B) and an increased occurrence ofmetastases (Fig. 1C). This predisposition to metastasis waseven more pronounced in patients with serum CD95L con-centrations �120 pg/mL (Fig. 1C). To further investigatewhether cl-CD95L is endowed with a prometastatic roletoward TNBC cells, we orthotopically transplanted lumines-cent TNBC cells (MDA-MB-231) in NOD/SCID/gc mice andmonitored both tumor growth and metastatic disseminationwith or without repeated injections of purified cl-CD95L. Ofnote, injection of cl-CD95L did not modify the tumor volume(Supplementary Fig. S1A), whereas as observed by biolumi-nescence imaging, it augmented the spread of TNBC cells,which seemed tometastasize to the lungs (Fig. 1D). To confirmthis observation, mice were sacrificed and luminescence wasassessed in different organs (Fig. 1E). Although no lumines-cence was detected in hearts and livers of control and cl-CD95L–injected mice (Supplementary Fig. S1B), lungs- andbrachial-draining lymph nodes experienced a dramatic TNBCcell invasion in mice injected with cl-CD95L as compared withcontrol mice (Fig. 1E). Overall, these findings indicated thathigh amounts of soluble CD95L mainly detected in TNBCwomen may participate in metastatic progression.

Next, to identify cells responsible for CD95L expression inpatients with breast cancer, immunohistochemistry was per-formed in breast tissue sections from healthy, patients withand without TNBC (Fig. 1F). CD95L staining was undetectablein healthy subjects, whereas breast tissues from patientswith and without TNBC exhibited a level of CD95L expres-sion correlated with serum concentrations (Fig. 1F). Indeed,CD95L was expressed in an increasing gradient from healthysubjects to patients with TNBC (Fig. 1F). Furthermore,CD95L was expressed in endothelial cells of blood vessels(CD31þ) surrounding tumor masses but not in the lymphaticendothelium (D2-40þ; Fig. 1G). These findings indicated thatCD95L is ectopically expressed in tumor blood vessels andits cleavage by metalloprotease releases a poor prognosticmarker associated with metastatic dissemination in patientswith TNBC.

Cleaved CD95L induces a p110b and Ca2þ-drivenpromotile signal in TNBC cells

Given that CD95L stems from blood vessels surroundingbreast cancer cells and is associatedwith the risk ofmetastasis,we next wondered whether this ligand contributed to TNBCintravasation by promoting cell motility. Different TNBC celllines exposed to cl-CD95L showed a dramatic increase in their

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Cancer Res; 73(22) November 15, 2013 Cancer Research6712







motility (Fig. 2A). Exposure to amounts of CD95L equal tothosemeasured in patients with breast cancer was sufficient totrigger cell motility. Indeed, titration of cl-CD95L showed thatcl-CD95L at 100 pg/mL, a dose corresponding to the meanconcentration measured in patients with TNBC (98.94� 45.37pg/mL; Fig. 1A), induced migration of TNBC cells (Supple-mentary Fig. S1C). To confirm the effect of soluble CD95L onpromoting the motility of breast cancer cells, we incubatedTNBC cells in the presence of sera from healthy donors orpatients with TNBC and evaluated cell migration using Boydenchambers. The results showed that the sera from women withTNBC, unlike that from healthy subjects, contained a moleculethat enhanced the migration of breast cancer cells (Fig. 2B).

This promotile factor was identified as soluble CD95L becausepreincubation of TNBC sera with a neutralizing anti-CD95Lantibody abrogated their promotile effect (Fig. 2B).

Activation of the PI3K signaling pathway promotes differentsteps leading to metastasis, including cell motility and intra-vasation/extravasation (13). PI3Ks are lipid kinases generatingthe second-messenger phosphatidylinositol (3,4,5)-trispho-sphate (PIP3), which serves as a docking site for varioussignaling factors. For instance, binding of the serine–threoninekinase Akt to PIP3 leads to its redistribution to the plasmamembrane, where it undergoes phosphorylation and activa-tion at Ser473 (for review, see ref. 14). In all tested TNBC cells,cl-CD95L induced rapid phosphorylation of Akt at Ser473

Figure 1. Serum CD95L is increased in TNBCwomen and is associated with metastatic dissemination. A, serum CD95L in patients diagnosed with or withoutTNBC was measured by ELISA (n, the number of patients per group). ��, P � 0.0001; ��, P � 0.01, by a two-tailed Student t test. B, Kaplan–Meierrelapse-free survival curves comparing patients with CD95L concentrations higher (thick line) or lower (dotted line) than 80 pg/mL. C, left, Kaplan–Meieranalysis of distant metastasis in patients with CD95L concentrations higher (thick line) or lower (dotted line) than 80 pg/mL. Right, Kaplan–Meier analysis ofdistantmetastasis in patientswith CD95L concentrations higher (thick line) or lower (dotted line) than 120 pg/mL. D, luciferase-expressingMDA-MB-231 cellswere orthotopically injected in NOD/SCID/gc null mice. Then, cl-CD95L (10 mg/kg) or vehicle was repeatedly injected and metastatic disseminationof TNBC cells was evaluated by bioluminescence imaging at day 21. The black arrows depict mice showing luciferase-expressing MDA-MB-231 spreading.E, mice treated as depicted in D were sacrificed at day 34. Intensity of luminescence was quantified in indicated organs. �, P < 0.05;��, P < 0.01, calculatedusing a two-tailed Mann–Whitney test. F, CD95L immunohistochemical staining of breast tissues from patients with the indicated breast cancer subtype(magnification, �20). The inset shows a �40 magnification of blood vessels. Stars indicate the tumor mass and black arrows show the blood vessels.G, immunohistochemical staininganalysis of consecutive sections of TNBC tissues stainedwithmarkersof blood (CD31þ) or lymphatic (D2-40þ) endothelium,and CD95L. The white arrowheads show a lymphatic vessel and black arrows depict a blood vessel.

Prometastatic Role of CD95L

www.aacrjournals.org Cancer Res; 73(22) November 15, 2013 6713







(Supplementary Fig. S2A). Furthermore, inhibition of the PI3Ksignal using the pharmacologic inhibitor LY294002 abrogatedthe migration of TNBC cells exposed to cl-CD95L (Supplemen-tary Fig. S2B), indicating that after cleavage by metallopro-tease, this ligand acts as a chemoattractant for malignantbreast cells through activation of PI3K. Class I PI3Ks comprisefour catalytic isoforms (a, b, d, and g). P110a and -b areubiquitously expressed, whereas expression of p110d and -gis restricted to hematologic cells. To evaluate the contributionof p110a and -b in CD95-mediated cell motility in TNBC cells,we analyzed the effect of pharmacologic inhibitors of p110a

and -b (Supplementary Fig. S2C). The p110b-inhibitor TGX-221was muchmore efficient than a drug-targeting p110a (PIK-90)to prevent both Akt phosphorylation (Supplementary Fig. S2D)and cell migration (Supplementary Fig. S2E) in TNBC cellsexposed to cl-CD95L. To confirm these observations, RNAinterference was used to silence expression of p110a or -bisoform (Supplementary Fig. S2F). Although downregulation ofp110a did not alter CD95-mediated PI3K activation (Fig. 2C)and cell migration (Fig. 2D), silencing of p110b inhibited Aktphosphorylation (Fig. 2C) and cell motility (Fig. 2D). Thesefindings demonstrated that cl-CD95L selectively activates the

Figure 2. cl-CD95L induces cell migration through the activation of the p110b catalytic subunit of PI3K. A, cell migration of the indicated TNBC cells incubatedwith or without cl-CD95L (100 ng/mL) for 24 hours was assessed using the Boyden chamber assay. Migrating cells were fixed and stained by Giemsa.For each experiment, five images of random fields were acquired. A representative image is shown. Bars, 70 mm. B, left, MDA-MB-231 cells were incubatedwith serum from healthy donors (control) or different TNBC women (P#, patient) for 24 hours in Boyden chambers. Migrating cells were fixed and stained byGiemsa and a representative image is shown. Bars, 70 mm. Right, Giemsa-stained migrating cells were lysed and absorbance was measured at awavelength of 560 nm. Values represent themeans�SEMof three independent experiments. �,P < 0.05 as calculated using a two-tailedMann–Whitney test.C, MDA-MB-231 breast tumor cells infected with lentivirus encoding p110a- and p110b-targeting short hairpin RNA (shRNA) were stimulated in thepresence (15 minutes) or absence (0 minute) of cl-CD95L (100 ng/mL), and the levels of phosphorylated Akt and total Akt were analyzed by immunoblotanalysis. D, top, the motility of the cells described in C was assessed using the Boyden chamber assay. Bottom, migrating Giemsa-stained cellswere lysed and absorbance was measured at a wavelength of 560 nm. Values represent the means � SEM of three independently performed experiments.E, TNBC cells were incubated with 100 ng/mL of cl-CD95L for the indicated times and then lysed. CD95 was immunoprecipitated and the immunecomplex was resolved by SDS-PAGE and analyzed by Western blot analysis with the indicated antibodies. F, top, MDA-MB-468 cells transduced withlentivirus encoding the indicated shRNAs were incubated for 24 hours in the presence or absence (control) of cl-CD95L (100 ng/mL) and cell migration wasassessed using Boyden chambers. Bottom, quantitative analyses of cell migration as described in B. Values represent the means � SEM of threeindependently performed experiments. �, P < 0.05, as calculated using a two-tailed Mann–Whitney test.

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PI3K catalytic subunit p110b, which promotes cell migrationin TNBC cells.

Cl-CD95L triggers MISC formation in TNBC cellsTo decipher the initial events leading to p110b activation,

TNBC cell lines were stimulated with cl-CD95L followed byimmunoprecipitation of CD95. An analysis of the resultingimmune complex revealed that CD95 did not bind to FADDand caspase-8 but did recruit c-yes (Fig. 2E). In addition,downregulation of c-yes expression (Supplementary Fig.S2G) prevented both Akt phosphorylation (Supplementary Fig.S2H) and cell migration (Fig. 2F) in TNBC cells exposed to cl-CD95L.Calcium ions (Ca2þ) participate in cell signaling as a second

messenger that relies on intensity (cytosolic concentration),temporal parameters (i.e., duration and frequency), and spatiallocalization to trigger a variety of cellular responses. BecauseCa2þ plays a pivotal role in cell motility (15), we analyzedits impact on the signaling pathway induced by cl-CD95L inbreast cancer cells. In nonexcitable cells, Ca2þ responsesmainly occur through a biphasic signal caused by activationof inositol 1,4,5-trisphosphate (IP3) receptors and the release ofCa2þ from the endoplasmic reticulum followed by a sustainedCa2þ entry across the plasmamembrane (16). Recently, STIM1was identified as the endoplasmic reticulum–located Ca2þ

sensor that links endoplasmic reticulum depletion to activa-tion of the plasma membrane Ca2þ channel formed by Orai1subunits, allowing Ca2þ to enter the cell (17). This store-operated calcium (Ca2þ) entry (SOCE) plays pivotal roles inboth the replenishment of the endoplasmic reticulum storeand cell signaling (18). Pretreatment of breast cancer cells withthe Ca2þ chelator, BAPTA-AM or the pharmacologic inhibitorof SOC channels BTP2 inhibited both CD95-mediated PI3Kactivation and cell migration (Fig. 3A and B). To determinewhether Orai1 participated in CD95 signaling in TNBC cells,Orai1 expression was blocked using shRNAmir. As shownin Fig. 3C, downregulation of Orai1 did not alter the CD95-mediated Ca2þ mobilization from the endoplasmic reticulum,but it abolished the subsequent Ca2þ entry observed in TNBCcells stimulated with cl-CD95L. Of note, Orai1-driven Ca2þ

entry was essential for inducing PI3K activation and cellmigration (Fig. 3D and E). To further characterize the mech-anistic link between CD95 engagement by cl-CD95 and Ca2þ

signaling in breast tumor cells, the contribution of c-yes andp110b to the CD95-mediated Ca2þ response was examined.Silencing of c-yes abrogated CD95-mediated Ca2þ signaling(Fig. 3F). On the other hand, although Ca2þ signaling contrib-uted to PI3K activation (Fig. 3A), the opposite was not true, asp110b knockdown did not affect the CD95-mediated Ca2þ

response (Fig. 3F). These results indicated that c-yes occupiesa proximal position in the sequence of events, leading to theactivation of the Ca2þ/PI3K signaling pathway in breast tumorcells exposed to cl-CD95L.

ROS production initiates the CD95-mediatednonapoptotic signalBecause recent reports have shown that Src kinases can

behave as redox sensors promoting cell migration in leuko-

cytes (19), we investigated whether CD95 activates c-yesthrough the generation of ROS. Different TNBC cells exposedto cl-CD95L experienced a rapid increase in intracellular ROS(Supplementary Fig. S3A), which was blocked by pretreatmentwith reduced nicotinamide adenine dinucleotide phosphateoxidase (NADPHox) inhibitors, such as DPI and apocynin (Fig.4A). To determine the effect of ROS on c-yes activation, c-yeswas immunoprecipitated and its autophosphorylation status(hallmark of its activation) was evaluated by immunoblottingusing a phospho-Src family antibody, recognizing Src whenphosphorylated at tyrosine 416 and c-yes at an equivalent site(tyrosine 426). In the presence of cl-CD95L, TNBCcells undergorapid activation of c-yes that is inhibited by NADPHox inhi-bitors (Fig. 4B and Supplementary Fig. S3B). Also, pretreatmentofmammary cancer cells withDPI or apocynin inhibitedCD95-mediated activation of Akt (Supplementary Fig. S3C and S3D)and cell migration (Supplementary Fig. S3E). The Nox family isdefined by seven distinct catalytic isoforms, namely Nox1 to -5and Duox1 and Duox2. Although Nox1, -2, -3, and -4 areassociated with p22Phox, which is involved in their propermembrane targeting and activity, Nox5, Duox1, and Duox2 donot require p22phox for their activity (20).MISC analysis showedthat p22Phox was recruited after stimulation with cl-CD95L(Fig. 4C). In addition, downregulation of p22Phox (Supplemen-tary Fig. S3F) prevented activation of c-yes and Akt (Fig. 4D)and cell migration in TNBC cells exposed to cl-CD95L (Fig. 4Eand Supplementary S3G), supporting that association ofp22Phox with Nox1, -2, -3, or -4 orchestrated the CD95-mediatedROS production. To identify the Nox subunit responsible forROS generation in TNBC cells stimulated with cl-CD95L, wenext analyzed the recruitment of Nox1, -2, -3, or -4 to theMISC.As shown in Fig. 4F, only Nox3 was recruited in the MISC ofTNBC cells stimulated with cl-CD95L. In addition, althoughNox2 downregulation (Supplementary Fig. S3H) did not altercell migration in cl-CD95L–stimulated TNBC cells (Supple-mentary Fig. S3I), Nox3 silencing (Supplementary Fig. S3H)completely abrogated it (Supplementary Fig. S3I). Finally,pharmacologic and genetic inhibitions of NADPH oxidasecompletely abrogated the CD95-mediated Ca2þ response (Sup-plementary Fig. S3J), confirming that CD95-triggered ROSgeneration is a proximal event in the signaling cascade inducedby metalloprotease-cleaved CD95L in TNBC cells. These find-ings highlighted that TNBC cells exposed to cl-CD95L undergoa rapidNox3-driven ROSproduction, resulting in the activationof c-yes and its downstream signaling pathway.

EGFR is essential for cl-CD95L–mediated migration ofbreast cancer cells

Taken together, these findings raise the question of howPI3K can be activated in TNBC cells in the presence of cl-CD95L. An in silico analysis of short linear motifs (21) in theintracellular region of CD95 did not reveal any consensussequences that could account for recruitment of p110b or itsregulatory subunit p85 after "death receptor" phosphorylationby c-yes, suggesting that at least one additional factor connectsCD95 to PI3K signaling. The Src kinase family has beenreported to phosphorylate EGFR at Tyr845, a modificationthat stabilizes the activation loop and maintains the receptor









in an active state (22). Moreover, Src kinase can also beassociated with EGFR to form a heterocomplex in TNBC cells(23). Because 72% of TNBC cells express EGFR (24) and itsexpression is correlated with cell migration (25), we examinedthe potential contribution of EGFR to CD95 signaling. EGFRwas expressed predominantly in TNBC cell lines comparedwith non-TNBC cells (Supplementary Fig. S4A and S4B). On thebasis of our immunoprecipitation experiments showing aninteraction between CD95 and c-yes (Fig. 2E), we hypothesizedthat recruitment of c-yes toMISCmay lead to EGFR activation,which in turn may serve as a molecular platform to elicit thePI3K (p110b) signaling pathway. To explore this hypothesis,the phosphorylation status of EGFR at Tyr845 was monitored.

As shown in Fig. 5A, exposure of MDA-MB-468 breast cancercells to cl-CD95L resulted in phosphorylation of EGFR atTyr845, which reached a peak at 2 minutes and disappearedafter 10 minutes of stimulation. This EGFR phosphorylationpreceded Akt activation (Fig. 5A). EGFR activation relied onCD95 stimulation because preincubation of TNBC cells withneutralizing anti-CD95L antibody (Nok-1) totally abrogatedEGFR phosphorylation (Supplementary Fig. S4C). In addition,EGFR phosphorylation was dependent on c-yes, as shown bythe loss of EGFR phosphorylation in TNBC cells in which c-yesexpression was suppressed (Fig. 5B). Next, we used biochem-ical and imaging approaches to elucidate whether EGFR wasrecruited to the MISC in TNBC cells exposed to cl-CD95L.

Figure 3. cl-CD95L evokes an Orai1-driven Ca2þ entry contributing to cell migration. A, MDA-MB-468 cells were preincubatedwith 5 mmof BAPTA-AM or 500ng/mL of BTP2 for 30 minutes and then treated or untreated (control) with 100 ng/mL cl-CD95L. Cells were then lysed and 100 mg of protein was loaded perlane. Immunoblot analysis was performed for phospho-ser473 Akt and total Akt. B, MDA-MB-468 cells were preincubated with 5 mmol/L BAPTA-AM or500 ng/mL BTP2 for 30 minutes and then treated or untreated (control) with 100 ng/mL cl-CD95L for 24 hours. Cell migration was assessed usingthe Boyden Chamber assay and the amount of Giemsa-stained migrating cells was quantified by measuring absorbance (O.D. 560 nm). Values representthe means � SD of three independent experiments. ��, P < 0.01, as calculated using a two-tailed Mann–Whitney test. C, MDA-MB-468 cells wereinfected with lentivirus encoding scrambled or Orai1-targeting shRNAs. The inset shows the immunoblot analysis of Orai1 silencing in transducedMDA-MB-468 cells. Tubulin was used as a loading control. Infected MDA-MB-468 cells were loaded with the Ca2þ probe Fluo8. The cells were then stimulatedwith 100 ng/mL cl-CD95L (black arrow) and [Ca2þ]i was monitored via the ratio F/F0 (relative Ca2þ[CYT]). Data represent mean � SD of F/F0 measuredin n cells. D, the cells depicted in Cwere stimulated for the indicated timeswith 100 ng/mLof CD95L and the activation status of Akt (phosphorylation at serine473) was evaluated by immunoblotting. E, MDA-MB-468 cells infected with lentivirus encoding scrambled or Orai1-targeting shRNAs were incubatedfor 24 hours in the presence or absence of cl-CD95L, and cell migration was assessed using Boyden Chamber assays. Migrating cells were stained (Giemsa)and lysed, and the percentage of migrating cells was quantified by measuring absorbance (OD 560 nm). F, the cl-CD95–triggered Ca2þ response wasmonitored in MDA-MB-468 cells in which c-yes (left) or p110b (right) expression was suppressed by RNA interference. Cells were preloaded with Fluo8 andthen stimulated with 100 ng/mL of cl-CD95L, and [Ca2þ]i was monitored. Data represent the mean � SD of the Ca2þ response in n cells.

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Immunoprecipitation of CD95 or EGFR from TNBC cellsexposed to cl-CD95L revealed the rapid formation of animmune complex containing CD95/c-yes/EGFR/p110b (Fig.5C and D). These biochemical observations were confirmedby the colocalization of EGFR and CD95 at the leading edge ofemitted pseudopodia in TNBC cells (Supplementary Fig. S4D

and S4E). Overall, these findings demonstrated that EGFR isrecruited to the MISC in TNBC cells exposed to cl-CD95L. Tofurther investigate the role of EGFR in the CD95-mediated cellmotility, we used the EGFR inhibitor erlotinib. Erlotinib pre-vented CD95-mediated Akt phosphorylation in TNBC cells(Supplementary Fig. S5A and S5B). In addition, erlotinib

Figure 4. Nox3-driven ROS production is instrumental in the CD95-mediated unconventional signaling pathway. A, MDA-MB-231 cells were loaded withthe ROS probe H2FDA and preincubated for 30 minutes with the NADPH oxidase inhibitors DPI (1 mmol/L) or apocynin (100 mmol/L) or dimethylsulfoxide (DMSO; control). Cells were then stimulated for 30 minutes with 100 ng/mL of cl-CD95L. ROS production was analyzed by flow cytometry.B, MDA-MB-231 cells were preincubated for 30 minutes with DPI (1 mmol/L) or apocynin (100 mmol/L) and then stimulated in the presence or absenceof cl-CD95L (100 ng/mL). C-yes was immunoprecipitated and its phosphorylation was assessed by immunoblotting. C, MDA-MB-231 cells wereincubated with 100 ng/mL of cl-CD95L for the indicated times and then lysed. CD95 was immunoprecipitated and the immune complex was resolved bySDS-PAGE and analyzed by Western blotting with the indicated antibodies. D, MDA-MB-231 cells were infected with lentivirus encoding scrambled orthree different p22Phox ShRNAs. These cells were treated with cl-CD95L (100 ng/mL) for indicated times, lysed, and 100 mg of protein per lane wasresolved by 10% SDS-PAGE. The activation status of Akt and the Src kinase family was monitored by immunoblotting for the detection ofphosphorylation at Ser473 and at Tyr426, respectively. Total protein is used as loading control. Data are representative of three independentexperiments. E, cell migration was assessed on the cells described in D in the presence or absence of cl-CD95L (100 ng/mL) for 24 hours using theBoyden chamber assay. Images are representative of three independent experiments. F, MDA-MB-231 cells were incubated with 100 ng/mL ofcl-CD95L (MISC) or APO1-3 (DISC) for the indicated times and then lysed. CD95 was immunoprecipitated and the immune complex was resolved bySDS-PAGE and analyzed by Western blotting with the indicated antibodies.









abolished migration of TNBC cells exposed to cl-CD95L (Sup-plementary Fig. S5C). To prove that EGFR was instrumental inCD95-mediated cell signaling, the tyrosine kinase receptor wasnext silenced (Supplementary Fig. S5D). Downregulation ofEGFR expression inhibited both CD95-mediated PI3K activa-tion (Fig. 5E) and cell migration in TNBC cells (Fig. 5F andSupplementary Fig. S5E). Examination of the role of EGFR in

the cl-CD95L–induced Ca2þ response showed that althoughthe binding of EGF to EGFR evoked aCa2þ response, whichwasblocked by erlotinib (Supplementary Fig. S5F), treatment withneither erlotinib nor EGFR silencing altered the CD95-medi-ated Ca2þ response (Supplementary Fig. S5F). This latterobservation questioned the involvement of EGF in the recruit-ment and activation of EGFR in breast cancer cells stimulated

Figure 5. CD95-dependent EGFR activation implements the PI3K signaling pathway. A, MDA-MB-468 cells were stimulated for the indicated times with cl-CD95L (100 ng/mL) and cells were then lysed. Phosphorylation of EGFR at Tyr845 and of Akt at Ser473 was monitored. Total EGFR and Akt servedas loading controls. B, MDA-MB-468 cells infected with lentivirus encoding c-yes shRNAs were stimulated for the indicated times with 100 ng/mL cl-CD95Land the cells were then lysed. The amounts of EGFR phosphorylation at Tyr845 and total EGFR were assessed by immunoblotting. C, MDA-MB-468cells were stimulated for the indicated times with cl-CD95L (100 ng/mL), lysed, and CD95 was immunoprecipitated. The immune complex was resolved bySDS-PAGEandanalyzedby immunoblottingwith the indicatedantibodies. Total lysatewasusedas input. D,MDA-MB-468cellswere stimulated as inC.Cellswere lysed and EGFR was immunoprecipitated. The immune complex was subjected to a SDS-PAGE and analyzed by immunoblotting. Total lysatewas used as input. Data are representative of three independent experiments. E, MDA-MB-231 cells infected with lentivirus encoding two differentEGFR-targeting shRNAswere incubatedwith (15) orwithout (0) cl-CD95L (100 ng/mL) for the indicated times and then lysed. Equal amounts of protein (100mg)were separated by SDS-PAGE and Akt phosphorylation at Ser473 was monitored by immunoblotting. Total Akt served as a loading control. F, cell migrationwas assessed in MDA-MB-231 cells described in E using the Boyden chamber assay. Data represent the means � SD of three independent experiments.

Malleter et al.




0 5 20 50 min

C-yes

CD95

EGFR

cl-CD95L

IP:C

D95

C

7255

40

Lys

ates

7255

170

D

7255

40

72

55

p110β

c-yes

EGFR

Lys

ates

0 5 20 50 min

CD95

IP:E

GF

R

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CD95

EGF-R

p110β

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EGFR

CD95

170

cl-CD95LA0 2 5 10 15 30 min

EGFRY845-P170130

EGFR170130

cl-CD95L

AktS473-P

Akt

7055

7055

BScr #1 #2

0 5 15 0 5 15 0 5 15 min

EGFR

EGFRY845-P

p110β

p110β130

130

F

Scr

EGFR sh#1R

elat

ive

nu

mb

er o

f m

igra

tin

g c

ells

(O

D56

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E

0 15 0 15 0 15 min

Scr #1 #2

7055

7055

Sh-EGFR

EGFR sh#2

Control cl-CD95L

AktS473-P

Akt

3

0

1

2

Scrambled sh#1

Control cl-CD95L

sh#2

Sh-EGFR

****

170130

170130

sh-c-yes





with cl-CD95L. To address the possible involvement of EGF inthis process, cl-CD95L–mediated signaling was examined inTNBC cells preincubated with cetuximab, an antibody thatbinds to the EGFR ectodomain and prevents its interactionwith EGF (26). Although this blocking antibody abrogated theEGF-induced Ca2þ response, it did not affect CD95-mediatedAkt activation (Supplementary Fig. S5G) and cell motility(Supplementary Fig. S5H), supporting the notion that CD95-driven recruitment of EGFR occurs through an EGF-indepen-dent mechanism in TNBC cells.

DiscussionThe results of the present study show that an elevated serum

concentration of CD95L in women diagnosed with breastcancer is associated with poor prognosis and high risk ofdistant metastasis. Furthermore, the distribution of CD95Lon endothelial cells of blood vessels suggests that its sheddingby a yet unknown metalloprotease may create the concentra-tion gradient required to promote intravasation of breasttumor cells and thus distant cancer metastases.Further analysis of the molecular pathway connecting the

"death receptor" CD95 to downstream PI3K signaling in TNBCcells exposed to cl-CD95L showed the recruitment and acti-

vation of EGFR. Although EGFR was necessary for CD95-mediated activation of PI3K, it was dispensable in the Ca2þ

response, indicating that the two pathways diverge early in theunconventional pathway of cl-CD95L–triggered signaling. Thisdivergence must occur downstream of ROS generation andsubsequent c-yes activation and upstream of EGFR activation,as inhibition of NADPH oxidase and silencing of c-yes expres-sion abolished both PI3K activation and Ca2þ signaling inTNBC cells exposed to cl-CD95L. In the presence of EGF, TNBCcells evoked a Ca2þ response that was abolished by pretreat-ment with the tyrosine kinase inhibitor erlotinib. Given thaterlotinib and EGFR downregulation did not alter the Ca2þ

signal observed in breast cancer cells exposed to cl-CD95L, wesurmise that recruitment and activation of EGFR by CD95occurs through an atypical mechanism independent of EGF.Confirming this assumption, the EGFR neutralizing antibodycetuximab did not impair CD95-driven EGFR activation inbreast cancer cells. Conventionally, binding of EGF ligands toEGFR leads to receptor dimerization, activation of its intrinsictyrosine kinase activity and subsequent phosphorylation ofdownstream signaling molecules. However, this dogma hasbeen challenged by the discovery that the activation of EGFR-mediated signaling can occur in a ligand-independent manner

Figure 6. CD95-mediated motilitysignaling pathway.









in the presence of ROS (27). In addition, G protein–coupledreceptor activation canmediate EGFR transactivation throughSrc family tyrosine kinases (28), and c-Src itself is able tofacilitate EGFR activation by phosphorylation of Tyr845 (22).Our results bring to light a novel mechanism for EGFR acti-vation in TNBC cells stimulated with metalloprotease-cleavedCD95L. From a mechanistic standpoint, CD95-driven EGFRactivation is dependent on the generation of ROS by Nox3. ROSactivate c-yes, which in turn recruits and activates EGFR.

Similar to EGFR, c-Met is a transmembrane receptor tyro-sine kinase for hepatocyte growth factor. Recent reports haveshown that theCD95 signaling pathway can bemodulated by c-Met (29). Indeed, the amino-acid residues YLGA in c-Met canbind CD95 (29) and prevent its homotrimeric self-aggregation,a pivotal step in the implementation of the apoptotic signal(30). It is noteworthy that the YLGAmotif is not detected in theEGFR sequence, and although the CD95/c-Met association islost when CD95L interacts with its receptor (31), binding of cl-CD95 is mandatory for recruitment of EGFR by CD95. Takentogether, these findings strongly suggest that receptor tyrosinekinases such as c-Met and EGFR can interact with CD95 bydifferent molecular mechanisms. More importantly, theseresults indicate that implementation of CD95-mediated non-apoptotic signals may occur through the recruitment of tyro-sine kinase receptors that behave as docking sites for severalproteins, including PI3K.

Because serum CD95L level is associated with relapse inbreast cancers, exhaustive identification of the components ofthe MISC and characterization of the molecular pathwayleading to the induction of CD95-mediated nonapoptoticsignaling are critical to understand how this "death receptor"can transmit non-death signals depending on the ligand withwhich it interacts. Our findings point to the activation of theSrc kinase c-yes, which implements Orai1-mediated SOCE,recruits EGFR, and induces the downstream activation of thep110b catalytic isoform of PI3K (summarized in Fig. 6).

In summary, this study identified serum CD95L level as anew prognostic marker of metastatic dissemination in womenwith breast cancer. This finding may help to guide clinicians inthe selection of the most appropriate treatment regimen forpatients with high levels of cl-CD95L who should undergointensive therapy, whereas patients with low cl-CD95L shouldbe spared. In addition, the elucidation of this atypical CD95-

mediated signaling pathway provides new therapeutic targetsfor preventing metastatic dissemination in TNBC. Althoughinhibitors of EGFR kinase activity may be attractive thera-peutic agents, especially because gefitinib and erlotinib arealready in use for the treatment of non–small cell lungcancer, most patients on prolonged gefitinib and erlotinibtreatment develop secondary mutations in the EGFR kinasedomain that block drug binding, leading to clinical resis-tance (32, 33). Therefore, inhibition of the CD95/CD95Linteraction may be a more appropriate treatment approach,especially because such an inhibitor already exists and iswell tolerated by patients (34).

Disclosure of Potential Conflicts of InterestM. Campone has honoraria from speakers' bureau and is a consultant/

advisory board member of Novartis. No potential conflicts of interest weredisclosed by the other authors.

Authors' ContributionsConception and design: M. Malleter, S. Tauzin, R. Castellano, Y. Collette,P. Vacher, P. LegembreDevelopment of methodology: M. Malleter, S. Tauzin, A. Bessede, R. Castel-lano, A. Goubard, L. Debure, P. VacherAcquisition of data (provided animals, acquired and managed pati-ents, provided facilities, etc.): M. Malleter, S. Tauzin, R. Castellano,A. Goubard, F. Godey, J. Leveque, P. J�ez�equel, M. Campone, T. Ducret,G. MacGrogan, P. VacherAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): M. Malleter, S. Tauzin, R. Castellano, L. Campion,M. Campone, P. Vacher, P. LegembreWriting, review, and/or revision of the manuscript:M. Malleter, S. Tauzin,L. Campion, M. Campone, Y. Collette, P. LegembreAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): A. Bessede, F. Godey, P. J�ez�equelStudy supervision: Y. Collette, P. Legembre

AcknowledgmentsThe authors thank A.M. Vacher and V. Velasco (Institut Bergoni�e, Bordeaux),

O. Cabaud, TrGET and animal facilities (CRCM, Marseille), and L3 and micros-copy facilities (SFR Biosit, Rennes) for their technical assistance.

Grant SupportThis work was supported by INCa, Ligue Contre le Cancer (Comit�es d'Ille-

et-Vilaine/du Morbihan/des Cotes d'Armor/du Maine et Loire et des Landes),ARC, Canc�eropole GO, R�egion Bretagne, R�egion Pays de la Loire, and RennesM�etropole.

The costs of publication of this article were defrayed in part by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received June 25, 2013; revised August 18, 2013; accepted September 3, 2013;published OnlineFirst September 26, 2013.

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CANCER RESEARCH | CORRECTION

Correction: CD95L Cell Surface CleavageTriggers a Prometastatic Signaling Pathwayin Triple-Negative Breast CancerMarine Malleter, S�ebastien Tauzin, Alban Bessede, R�emy Castellano,Armelle Goubard, Florence Godey, Jean Leveque, Pascal J�ez�equel,Loic Campion, Mario Campone, Thomas Ducret, Ga€etan MacGrogan,Laure Debure, Yves Collette, Pierre Vacher, and Patrick Legembre

In the original version of this article (1), theWestern blot image used in Fig. 5D to representCD95 lysate was inadvertently duplicated from the image of c-yes lysate. The image hasbeen replaced with the intended image for CD95 lysate. In addition, in SupplementaryFig. S5H MDA-MB-468 cells, the cell migration images for control and cl-CD95L in theuntreated group were inadvertently duplicated from the control group of SupplementaryFig. S5C MDA-MB-231 cells. The images have been replaced with the intended images forMDA-MB-468 cells. These corrections are reflected in the latest online HTML and PDFversions of the article. The authors regret these errors.

Reference1. Malleter M, Tauzin S, Bessede A, Castellano R, Goubard A, Godey F, et al. CD95L cell surface cleavage

triggers a prometastatic signaling pathway in triple-negative breast cancer. Cancer Res 2013;73:6711–21.

Published online February 3, 2020.Cancer Res 2020;80:639doi: 10.1158/0008-5472.CAN-19-3636�2020 American Association for Cancer Research.

AACRJournals.org | 639

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2013;73:6711-6721. Published OnlineFirst September 26, 2013.Cancer Res Marine Malleter, Sébastien Tauzin, Alban Bessede, et al. Signaling Pathway in Triple-Negative Breast CancerCD95L Cell Surface Cleavage Triggers a Prometastatic

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