Therapeutics, Targets, and Chemical Biology

A Cell-Penetrating Peptide Targeting AAC-11Specifically Induces Cancer Cells DeathL�eonard Jagot-Lacoussiere1,2, Ewa Kotula1,2, Bruno O. Villoutreix2,3,4,Heriberto Bruzzoni-Giovanelli1,2,5, and Jean-Luc Poyet1,2,4

Abstract

AAC-11 is an antiapoptotic protein that is upregulated in mostcancer cells. Increased expression of AAC-11 confers a survivaladvantage when cancer cells are challenged with various stressesand contributes to tumor invasion and metastases, whereas itsderegulation reduces resistance to chemotherapeutic drugs. Theantiapoptotic effect of AAC-11 may be clinically relevant as itsexpression correlates with poor prognosis in several humancancers. Thus, inactivation of AAC-11 might constitute an attrac-tive approach for developing cancer therapeutics. We have devel-oped an AAC-11–derived cell-penetrating peptide, herein namedRT53, mimicking in part the heptad leucine repeat region ofAAC-11, which functions as a protein–protein interactionmodule, and that can prevent AAC-11 antiapoptotic properties.In this study, we investigated the anticancer effects of RT53. Ourresults indicate that RT53 selectively kills cancer cells while

sparing normal cells. RT53 selectively inserts into the mem-branes of cancer cells, where it adopts a punctate distributionand induces membranolysis and release of danger-associatedmolecular pattern molecules. Systemic administration of RT53inhibited the growth of preexisting BRAF wild-type and V600Emutant melanoma xenograft tumors through induction ofapoptosis and necrosis. Toxicological studies revealed thatrepetitive injections of RT53 did not produce significant tox-icity. Finally, RT53-killed B16F10 cells induced tumor growthinhibition in immunocompetent mice following a rechallengewith live cancer cells of the same type. Collectively, our datademonstrate that RT53 possesses tumor-inhibitory activity withno toxicity in mice, suggesting its potential as a therapeuticagent for the treatment of melanoma and probably othercancers. Cancer Res; 76(18); 5479–90. �2016 AACR.

IntroductionAAC-11 (antiapoptosis clone 11), also called Api5 or FIF, is an

antiapoptotic protein whose expression prevents apoptosis fol-lowing growth factor deprivation (1). Many cancer cells exhibitelevated levels of AAC-11, which were found to correlate withpoor prognosis in patients with non–small cell lung cancer andcervical cancer and contribute to tumor invasion and metastases(2–10). Interestingly, AAC-11 appears to be involved in antican-cer drugs-induced apoptosis of tumor cells. Indeed, we haveshowed that AAC-11 gene silencing remarkably decreased che-moresistance,whereas its expression interferedwithdrug-inducedapoptosis (11). Increased expression of AAC-11was also found toprotect from UV-induced apoptosis whilst its depletion is cancer

cells lethal under condition of low-serum stress (10, 12).Although the precise mechanisms by which it suppresses apopto-sis remain unclear, AAC-11 is now known to interfere with bothE2F1-mediated apoptosis and Acinus-dependent apoptotic DNAfragmentation (11, 12). Finally, AAC-11 has been recently dem-onstrated to confer tumor immune resistance to antigene-specificT cells (13). These observations make AAC-11 a significant playerin cancer cell progression and survival.

AAC-11 interacts with several apoptosis-related proteins andthis complex-forming ability, probably favored by its elongated3D structure (14), appears to be essential for AAC-11 to fulfillits antiapoptotic functions (1, 9, 11, 15, 16). Therefore, inhi-bitors of AAC-11 protein–protein interactions could prove tobe of clinical benefit. We have developed a cell-penetratingpeptide spanning the heptad leucine repeat region domain ofAAC-11 (residues 363–399) fused at the N-terminus to thetransmembrane-penetrating sequence penetratin. This peptidewas able to disrupt the endogenous AAC-11–Acinus complexand drastically increase drug-induced cytotoxicity in a varietyof cancer cells (11). Here, we characterized the anticancerproperties of this AAC-11 targeting peptide, herein namedRT53. We find that RT53 induced plasma membrane permea-bilization of cancer cells while sparing nonmalignant cells.Interestingly, RT53 was able to inhibit tumor growth in vivoin human melanoma xenograft models, BRAF wild-type andV600E mutant, without systemic toxicity. Moreover, RT53-treated mouse melanoma cells mediated anticancer effects ina tumor vaccination model. These findings provide support forthe use of AAC-11-inactivating peptides as a novel treatmentstrategy for melanoma and possibly other cancers.

1INSERM UMRS1160, Institut Universitaire d'H�ematologie, HopitalSaint-Louis, Paris, France. 2Universit�e Paris Diderot, Sorbonne ParisCit�e, Paris, France. 3INSERM UMRS 973, Paris, France. 4c-Dithem,Inserm Consortium for Discovery and Innovation in Therapy andMedicine. 5Centre d'Investigations Cliniques 9504 INSERM-AP-HP,Hopital Saint-Louis, Paris, France.

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

L. Jagot-Lacoussiere and E. Kotula contributed equally to this article.

Corresponding Author: Jean-Luc Poyet, INSERM U1160, Universit�e ParisDiderot, Sorbonne Paris Cit�e, Institut Universitaire d'H�ematologie, HopitalSaint-Louis, 1 rue Claude Vellefaux, 75010 Paris, France. Phone: 33-1-42499263; Fax: 33-1-42385345; E-mail: jean-luc.poyet@inserm.fr

doi: 10.1158/0008-5472.CAN-16-0302

�2016 American Association for Cancer Research.

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Materials and MethodsPeptides

Peptides were synthesized by Proteogenix (Strasbourg, France)and were >95% pure as determined by HPLC and mass spectro-graphic analysis.

Cell lines, apoptosis, cytochemistry andcoimmunoprecipitation assays

A375, C8161, COLO 792, MEWO, Lu1205, and SK-Mel-28cells were provided by Dr. N. Dumaz (INSERM U976) andgenotyped to verify their authenticity. MRC-5, B16F10 andH1299 cells were provided by Drs. M. Dutreix (CNRS UMR3347,INSERM U1021) and R. Fa

�hraeus (INSERM U1162), and were

purchased from ATCC. HaCat cells were provided by Prof. N.Basset-Seguin (AP-HP, Hôpital Saint-Louis, Paris, France) andtheir characteristics were described elsewhere (17). A549, MCF7,HeLa, THP-1, and KARPAS 299 were purchased from The Euro-pean Collection of Cell Cultures. SU-DHL-5 cells were purchasedfrom ATCC. Cells were cultivated either in DMEM or RPMI 1640(Life Technologies), supplemented with 10% FBS and 1% pen-icillin/streptomycin. Apoptosis, immunoprecipitations, Westernblot, and cytochemistry analysis were performed as previouslydescribed (18, 19).

Materials and antibodiesAll chemicals were purchased from Sigma. Antibodies were

from Santa Cruz Biotechnology (mouse anti-Acinus, rabbit anti-AAC-11), Cell Signaling Technology (rabbit anti-Acinus, rabbitanti-Cox IV, rabbit anti-cytochrome c, mouse anti-Smac/Diablo,and rabbit anti-HMGB1), and Thermofisher (mouse anti-AIF).

Cell viability, LDH release, ATP release, in vitro caspases assays,and HMGB1 release

Cells survival was assessed with the CellTiter 96 Aqueous OneSolution Cell Proliferation Assay kit (Promega). For Dym Assess-ment, cellswere stainedwith 100nmol/LDiOC6(3) and analyzedby flow cytometry. Release of lactate dehydrogenase (LDH) andATP in the culture medium were assessed with the CytoTox 96Non-Radioactive Cytotoxicity Assay and Enliten ATP Assay,respectively (Promega). Caspase-3/7 and caspase-9 activitiesmea-sured using the Caspase-Glo 3/7 and Caspase-Glo 9 assay systems(Promega), respectively. Data are means � SEM (n ¼ 3). ForHMGB1 release, cells supernatants were collected at the indicatedtimes and concentrated by Centricon (Millipore) 10-kDa filter.Cells were harvested and lysed in Laemmli sample buffer. Thelysates or equal volumes of the concentrated supernatants werethen analyzed by Western blotting.

Mitochondrial preparationHeLa cells were resuspended in buffer A (250 mmol/L sucrose,

20 mmol/L HEPES, 10 mmol/L KCl, 1.5 mmol/L MgCl2,0.5 mmol/L EGTA, and pH 7.5) and homogenized in a Douncehomogenizer. The homogenate was centrifuged twice 10minutesat 750� g. Supernatants were centrifuged 15minutes at 10.000�g and the resultingmitochondrial pellets resuspended in buffer A.The supernatants were further centrifuged at 100,000 � g for 1hour and the resulting supernatants (designed as S100) frozen at�80�C.

In vitro assay of mitochondrial proteins releaseHeLa cells mitochondria (10 mg) were incubated with RT53 or

RT53M in a final volume of 25 mL of buffer A for 1 hour at 30�C.The samples were then centrifuged (10,000 � g, 10 minutes) topellet the mitochondria. The resulting supernatant and pelletswere fractionated by SDS-PAGE followed by immunoblotting.

Measurement of mitochondrial uptake of Rhodamine-labeledRT53 or RT53M in isolated mitochondria

HeLa cellsmitochondria (10mg)were incubatedwith 5mmol/Lof Rhodamine-labeled RT53 or RT53M for 1 hour at 30�C.Uptakewas stopped by centrifugation (10,000 � g, 10 minutes), themitochondrial pellet was washed twice in buffer A and resus-pended in PBS containing 0.2% Triton X-100. The solutioncontaining the mitochondria (100 mL) was transferred in a 96-well black plate and the fluorescence of rhodamine measured at555 nm (excitation, 580 nm).

shRNALentiviral particles were produced in HEK293 with the helper

plasmids pLvVSVg and pLvPack (Sigma) plus a lentiviral plasmid.shRNAs against AAC-11 originate from lentiviral plasmids MIS-SION pLKO.1-puro (Sigma; clone A: NM_006595.2-278s1c1,containing the sequence CCGGGCAGCTCAATTTATTCCGAAAC-TCGAGTTTCGGAATAAATTGAGCTGCTTTTTG and clone B:NM_006595.2-224s1c1, containing the sequence CCGGGCCTA-TCAAGTGATATTGGATCTCGAGATCCAATATCACTTGATAGGCT-TTTTG). shSCR (SHC016-1EA, Sigma) contains the sequenceCCGGCAACAAGATGAAGAGCACCAACTCGAGTTGGTGCTCTT-CATCTTGTTGTTTTTG. Lentiviral infection of target cells wasdone as previously described (19).

Structural analysis of the peptideSecondary structure predictions were performed with PSIPRED

(20) and JPred4 (21)while themeta serverMESSAwas used for in-depth sequence analysis (22). Three-dimensional structure pre-dictions were carried out with the PEP-FOLD server (23), andpredictions of orientation of the peptide in the membrane weredone with the PPM server (24). Figures were generated withPyMOL (http://www.schrodinger.com).

Mice toxicity studiesAnimal experiments were approved by The University Board

Ethics Committee for Experimental Animal Studies (#2303.01).Two groups of 3 nudemice were exposed to single (acute toxicity)or daily intraperitoneal (i.p.) administration for 5 consecutivedays (subacute toxicity) of increasing doses of RT53 in normalsaline or normal saline alone. For each treatment schedule, weightchange, signs of toxicity were monitored up to 14 days after thelast administration. Blood was collected from the saphenous veinbefore injections started and 2 hour following the last injectionand the samples analyzed for cell count using an automatedhematology analyzer (MedoniCA620, Stockholm, Sweden). Allmice were euthanized by cervical dislocation under anesthesia. Atthe moment of euthanasia, the organs were harvested and under-went extensive macroscopic and microscopic examination.

Immunogenicity assay of RT53 in miceMale FVB/Nmice were immunized with 125 mg of RT53 daily

for 5 weeks. Blood samples were collected before and 5 weeks

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after the first immunization, and antibodies were detectedusing an IgG mouse ELISA Kit (Abcam) assay against mousewhole IgG.

Xenograft tumor modelHuman melanoma xenograft tumors were obtained by subcu-

taneous (s.c.) injection of 4 � 106 of SK-Mel-28 or 2 � 106 ofC8161 cells (100 mL) in the right flank of 6-week-old nude mice(Centre-Elevage-Janvier). Mice were maintained in specific path-ogen-free animal housing (IUH). Treatment started after random-ization when tumors were visible and consisted of daily i.p.injection of normal saline solution or RT53 or RT53M in normalsaline solution (n ¼ 7 per group). Tumor growth was monitoredby a digital caliper and volume was calculated using the formula:Length x Width2/2. Animals were euthanized after 21 days oftreatment or when tumor size reached the ethical endpoint.

Ex vivo imaging and tissue distributionNude mice bearing subcutaneous C8161 human melanoma

xenografts were injected i.p. with either 5 mk/kg of rhodamine-labeled RT53 in normal saline or normal saline alone. The micewere euthanized 1hour after injection, and the organs and tumorswere isolated and the fluorescence observed by an IVIS spectrumin vivo imaging system (Caliper).

Histological analysis and TUNEL assayTumorswerefixed in 4%neutral buffered formalin and embed-

ded in paraffin. Sections (4 mm) were stained with hematoxylin-eosin (H&E) and subjected to microscopic analysis. Anti-Ki-67antibody (Abcam) was used as marker for tumor cell prolifera-tion. The incidence of apoptotic tumor cells was assessed byTUNEL assay with the In-Situ Cell Death Detection Kit (Roche).Histological analysis was performed at the HistIM facility ofCochin Institute (Paris, France). Slides were imaged using aLamina multilabel slide scanner (Perkin Elmer).

Tumor vaccination assayB16F10 cellswere exposed to30mmol/LRT53 for20hour for cell

death inductionand inoculated subcutaneously (2�106 cells) intothe left flanks of C57BL/6mice (n¼ 8 per group). Seven days later,the mice were challenged subcutaneously on the right flank with 1� 106 live B16F10 cells. Tumor growth on the challenge site wasevaluated using a digital caliper. Animals were euthanized whentumor size reached the ethical end point or were necrotic.

Statistical analysisThe Student t test was used to test for statistical significance of

thedifferences between thedifferent groupparameters.P values ofless than 0.05 were considered statistically significant.

ResultsRT53 induces cancer cells but not normal cells death in vitro

We previously demonstrated that preincubation of cancer cellswith a low dose of RT53 (see Fig. 1A) increases drug-induced celldeath and prevents AAC-11 interaction with the apoptotic factorAcinus (11). In line with our previous data (11), amutant peptide(RT53M, see Fig. 1A) in which leucines 384 and 391 (AAC-11numbering) were substituted by glycines did not sensitize cancercells to etoposide-mediated cell death nor it prevented AAC-11interaction with Acinus (Supplementary Fig. S1). We assessed the

viability of melanoma SK-MEL-28 cells or nonmalignant HaCaTcells following exposure to increasing concentration of RT53. Asshown in Fig. 1B, RT53 exhibited dose-dependent cell deathactivity in SK-MEL-28 cells, whereas very low toxicity wasobserved in HaCat cells. However, RT53M as well as the pene-tratin domain alone (see Supplementary Table S1) did notdecrease cell viability of the cancer cells (Fig. 1B). The effect ofRT53 on cellular viability was further assessed on various solidand hematological tumor cell lines, as well as normal cells. Asshown in Fig. 1C, RT53 exhibited cell death activity in all cancercells tested above 15 to 20 mmol/L in both BRAFV600E (SK-Mel-28,Lu1205, and A375) and BRAFWT (C8161, MEWO, and COLO792)melanoma cells. Interestingly, RT53was essentially not toxicto the normal cells tested (Fig. 1C). Here again neither RT53Mnorthe penetratin domain alone induced cell toxicity (data notshown). We next investigated the effect of the internalizationsequence upon cancer cell toxicity by conjugating the AAC-11(363-399) domain to the transactivator of transcription (TAT)cell-penetrating sequence (25). The resulting TAT-(363-399) pep-tide (Supplementary Table S1) was at least as effective as RT53 fordecreasing SK-MEL-28 cancer cells viability (Fig. 1D). Finally,attaching the penetratin sequence on the C-terminal had noinfluence on the cytotoxic properties of the AAC-11 (363-399)domain as the resulting (363–399)-Pen peptide (SupplementaryTable S1) elicited similar cytotoxicity as RT53 toward cancerouscells (Fig. 1D). Combined, these data indicate that the cytotoxiceffect of RT53 is not a nonspecific effect of the leader sequence.

RT53 causes caspase-independent and RIPK1-independentcell death

We next investigated RT53mechanisms of cancer cell death. Asshown in Fig. 2A, nontoxic concentrations of cycloheximide didnot alter RT53 cytotoxic action, suggesting that RT53-induced celldeath does not depend on de novo protein synthesis. Caspaseactivity assays indicated that RT53, but not RT53M, treatment ofA549 cells promoted caspase-9 as well as caspase-3/7 activation(Fig. 2B), albeit at relatively low levels compared to etoposide-induced cell death (not shown). However, treatment of cells withthe pan-caspase inhibitor zVAD-fmk failed to prevent RT53-induced cell death (Fig. 2C), suggesting that the observed cyto-toxicity of RT53 is not dependent on caspases.

Inhibition of receptor-interacting protein kinase 1 (RIPK1) bynecrostatin-1 did not inhibit RT53 cytotoxic activity, therebyexcluding necroptosis (Fig. 2D). Finally, 3-methyladenine failedto inhibit RT53-cytotoxic effect, thus excluding autophagic celldeath (Fig. 2E). Combined, our results indicate that RT53-medi-ated cell deathmechanism is independent of caspases andnecrop-tosis, as well as autophagy.

To determine whether AAC-11 availability was a necessaryprerequisite for the cytotoxic effect of RT53, AAC-11 expressionwas knocked down in SK-MEL-28 and C8161 cells by using twodifferent shRNAs, introduced through lentiviral particles. Asshown in Fig. 2F, silencing of AAC-11 did not have any significanteffect on RT53 cytotoxicity in SK-MEL-28 or C8161 cells, indi-cating that the cytotoxic effect of RT53 does not depend on AAC-11 expression levels.

RT53 induces cancer cells membrane lysisWe then evaluated necrotic cell death of SK-MEL-28 cells

treated with RT53 by propidium iodide (PI) uptake assay. As

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shown in Fig. 3A, SK-MEL-28 cells, but not HaCat cells, displayedan increasing loss of plasma membrane integrity rapidly afterexposure to RT53, whereas RT53M treatment did not induceplasmamembrane alteration. Similar results were obtained usingA549, C8161, MCF-7, Karpas-299, and SU-DHL-5 cell lines (datanot shown).

To monitor the effect of RT53 at the morphological level, weperformed time-lapse fluorescence imaging of C8161 cellsexposed to RT53 or RT53M in the presence of PI. Incubationwith RT53, but not RT53M, induced massive cell blebbing andswelling, together with PI incorporation and accumulation ofcellular debris after 3 hours of incubation (Fig. 3B).

Necrotic cells release endogenous molecules, such as LDH,high-mobility group box 1 (HMGB1), or ATP (26, 27). As shownin Fig. 3C, RT53 exposure resulted in a drastic increase in LDHrelease into SK-MEL-28 cell supernatants, indicating membranedamage, whereas RT53M-incubated cells or nonmalignant HaCatcells showed little LDH release. Release of LDH was concomitantwith PI permeability (Fig. 3B). RT53, but not RT53M, treatment ofSK-Mel-28 cells increased levels of extracellular HMGB1 (Fig. 3D)

and ATP (Fig. 3E). Combined, these data indicate that RT53induces rapid necrosis, via membranolysis, of cancerous cells.

To investigate whether RT53 targets the mitochondria for celldeath induction, we assessed mitochondrial transmembranepotential (Dym) by using the fluorescent potentiometric probeDiOC6(3), by flow cytometry. Significant loss of Dym wasobserved following RT53, but not RT53M, treatment, as indicatedby a decrease in DiOC6(3) intensity, indicating a breakdown ofmitochondrial membrane integrity (Fig. 3F). Necrosis is accom-panied by mitochondrial swelling and loss of mitochondrialmembrane potential (28). Therefore, the observedmitochondrialdamage following RT53 incubation could either constitute asubsequent event of RT53 intracellular action or be a side effectcaused by membranolysis-induced necrosis. To examine whetherRT53 induces mitochondrial release of apoptogenic factors, puri-fiedmitochondria fromHeLa cells were incubatedwith increasingamounts of RT53 or RT53Mwith or without cytosolic extracts. Asshown in Supplementary Fig. S2A, neither peptide induced anydetectable cytochrome c, AIF, and Smac release, as opposed to thecontrol atractyloside. We then assessed RT53 mitochondrial

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RT53 selectively induces cancer cells death. A, amino-acid sequence of RT53 and RT53M. The penetratin sequence is in bold. In RT53M, mutations (leucines toglycines) are underlined. B, viability of SK-MEL-28 or HaCaT cells exposed to increasing concentrations of penetratin domain (Penetratin), RT53, or RT53Mfor 20hours.C, the indicated cellswere exposed to increasing concentrations ofRT53 for 20hours.D,SK-MEL-28orHaCaT cells exposed to increasing concentrationsof RT53, TAT-(363-399), or (363-399)-Pen for 20 hours.

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uptake by incubating rhodamine-labeled RT53 or RT53M withisolated mitochondria. No fluorescence was detected from mito-chondria incubated with RT53 or RT53M, in contrast with theRhodamine 123 control (Supplementary Fig. S2B), indicatinglack of uptake of RT53 by isolated mitochondria. Combined,these results suggest that the oncolytic effect of RT53 mostlyinvolves plasma membrane perturbation, the observed mito-chondrial alterations likely being the consequence of the resultingnecrotic death.

Because of itsmembrane lytic activity,we studiedwhether RT53can bind to the plasma membrane. C8161 or HaCat cells wereincubated with rhodamine-labeled RT53 or RT53M and thepeptides respective fluorescent patterns analyzed. To avoid tox-icity, we used a 5 mmol/L sublethal concentration of RT53(see Fig. 1C). Strikingly, RT53 localized in a punctate pattern atthe plasmamembrane of the cancerous C8161 cells together witha diffuse intracellular distribution (Fig. 4A). This punctate pattern

suggests a compartmentalized accumulation for RT53 in mem-brane microdomains rather than nonspecific binding. Interest-ingly, RT53 distributed rather uniformly throughout the untrans-formed HaCa cells, without accumulation at the cell membrane.Finally, RT53Mpeptide exhibited a homogeneous pattern in bothtransformed and untransformed cells (Fig. 4A). Overall, theseobservations suggest that the discrete, punctate pattern observedfor RT53 in cancer cellsmembranes could be indicative of bindingof RT53 to cancer cells' membrane specific target(s). Because RT53is only cytotoxic to cancer cells, our data imply that the resultingmembrane sequestration of RT53 could be necessary for its cancercells membranolysis properties.

Although membrane-active peptides (MAP) exhibit signifi-cant variability in conformations, a number of MAPs possess alinear a-helical structure (29). To obtain structural informationabout RT53, we used two well-established secondary structureprediction servers: PSIPRED (20) and JPred4 (21) as well as the

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RT53 induces caspase-independent and RIPK1-independent cell death. A, viability of SK-Mel-28 cells exposed to 20 mmol/L of RT53 or RT53M in the presence orabsence of 10 mg/mL cycloheximide (CHX) for 20 hours. B, A549 cells exposed to increasing concentrations of RT53M or RT53 for 20 hours. Caspase-3/7and -9 activities were measured. C, viability of A549 cells exposed to 20 mmol/L of RT53 or RT53M in the presence and absence of 50 mmol/L zVAD-fmk for20 hours. Etoposide (25 mmol/L) was used as a control. D, viability of THP-1 cells exposed to 20 mmol/L of RT53 or RT53M in the presence and absence of 25 mmol/LNecrostatin-1 (Nec-1) for 20 hours. TNFa (30 ng/mL) þ zVAD-fmk (40 mmol/L) treatment was used as a control. E, viability of A549 cells exposed to20 mmol/L of RT53 or RT53M in the presence and absence of 10 mmol/L 3-methyladenine (3-MA) for 20 hours. Rapamycin (100 nmol/L) was used as a control.F, left, Western blot analysis of AAC-11 knockdown efficiency in stable clones infected with lentivirus-shRNAs AAC-11 (A and B) or scramble-shRNA (SCR). Right,viability of SK-MEL-28 or C8161 cells expressing the indicated shRNAs exposed to 20 mmol/L of RT53 or RT53M for 20 hours.

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MESSA meta server for in-depth sequence analysis and searchfor homologous structural templates (22). The secondary struc-ture prediction methods indicate that RT53 should essentiallyadopt an a-helical structure (not shown), in agreement withthe known 3D structures of the two parts of the peptide, thepenetratin segment (30) and the AAC-11 protein (14),yet with a possible, but less probable, break around the RT53peptide residues FARGL. Three-dimensional structure predic-tions carried out with the PEP-FOLD server (23) also suggestedan essentially helical structure for RT53 (Fig. 4B). We furtherexplored the possible orientation of the RT53 peptide with acell membrane with the PPM web server (24). Spatial posi-tions prediction analysis indicated that RT53 should belong tothe peripheral protein type as its predicted transfer energyfrom the solvent to the membrane would be around �8.6kcal/mol. Therefore, the peptide could have a surface orien-

tation where its long axis would be parallel to the membranedue to the partial amphipatic nature of the molecule (Fig. 4C,left). Yet, an alternative, probable transmembrane orientationis possible due to the presence of a relatively long, mainlyhydrophobic segment (residues FARGLQVYIRQL; Fig. 4C,right). Overall, these data suggest that RT53 appears to possessa membrane active conformation, which likely explains itspore-forming ability when retained in the membrane of cancercells.

RT53 treatment inhibited melanoma tumor growth inmelanoma mouse xenograft models

We next sought to explore if RT53 peptide might represent apossible therapeutic strategy to suppress tumor growth in vivo. Wechose to focus our study on melanoma because the clinicalmanagement of this highly aggressive skin cancer remains

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RT53 induces cancer cells membranolysis. A, SK-Mel-28 or HaCat cells were exposed to 20 mmol/L of RT53 or RT53M for the indicated times. Plasma membraneintegrity was assessed by flow cytometry using PI staining. B, C8161 cells were exposed to 20 mmol/L of RT53 or RT53M in the presence of 2.5 mmol/L PI. Cellmorphology was monitored by time-lapse microscopy. C, SK-Mel-28 or HaCat cells exposed to 20 mmol/L of RT53 or RT53M for the indicated times. ExtracellularLDH into the culture medium was measured. The obtained values were normalized to those of the maximum LDH released (completely lysed) control.D, SK-Mel-28 cells were left untreated or exposed to 20 mmol/L of RT53 or RT53M for the indicated times. Cell lysates (L) and culture supernatant (S) were analyzedby Western blot for HMGB1. E, SK-Mel-28 cells were exposed to 20 mmol/L of RT53 or RT53M for the indicated times, and extracellular ATP was measured in theculture medium. F, SK-Mel-28 cells were exposed to increasing concentrations of RT53 or RT53M for 20 hours. Dym was assessed by flow cytometryusing the fluorescent probe DiOC6(3).

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challenging (31). Preliminary toxicity analysis in nude miceindicated that neither repetitive i.p. administration of RT53 atdoses up to 60mg/kg nor single i.p. administration at doses up to100 mg/kg affected mouse behavior or growth (Fig. 5A). Nonoticeable changes in complete blood counts or organ toxicity(macroscopic or microscopic) were noted with either treatmentschedule (data not shown). Measure of the antibody responseusing immunocompetent mice did not reveal antibody produc-tion against RT53 after 5 weeks of daily injections (Fig. 5B).Combined, these observations indicate that RT53 exhibits limitedor nonexistent immunogenicity and toxicity in mice.

We next evaluated the efficacy of RT53 in both BRAFV600E (SK-Mel-28) and BRAFWT (C8161) xenograft models of melanoma.Interestingly, treatment with RT53, but not RT53M, inducedsignificant tumor growth inhibition in both C8161 (approximatetumor growth reduction of 63%, P < 0.005; Fig. 5C) and SK-Mel-28 (approximate tumor growth reduction of 80%, P < 0.005; Fig.5D) xenograft models as compared with saline-injected mice.These data demonstrated that RT53was able to reduce the growthof melanoma tumors at distant site as single agent and regardlessof their BRAF mutational status.

To better understand the in vivo performance of RT53, westudied the biodistribution of a rhodamine-labeled peptideadministrated i.p. in mice-bearing subcutaneous C8161 tumorsusing an IVIS imaging system (Xenogen). RT53 was detectable inthe liver, lungs, spleen, and kidneys, but not in the brain, suggest-

ing that it may not cross the blood–brain barrier (Fig. 6A). Veryinterestingly, accumulation of the fluorescent conjugate in thetumor was clearly evident, indicating that RT53 is able to reachand accumulate in tumors in vivo.

We next investigated the mechanism by which RT53 inhibitstumor growth by examining necrosis, cell proliferation, andapoptosis in time- and size-matched xenograft tumors gener-ated by RT53-treated or control groups. As shown in Fig. 6B(top), hematoxylin–eosin staining of tumor sections revealedlarger percentages of necrotic regions in RT53-treated tumorscompared with tumors from control groups. In non-necroticregions, no obvious differences in proliferation (Ki-67 staining)were seen between the different tumors (Fig. 6B, middle).Interestingly, an increased number of apoptotic cells wereobserved in RT53-treated tumors compared with controltumors (Fig. 6B, bottom). Combined, these data indicate thatRT53 inhibited melanoma tumor growth by inducing necrosisand triggering apoptosis.

RT53-treated B16 melanoma cells induce anticancer responseOur results indicate that RT53 can induce the release of

HMGB1, cytochrome c, and ATP from cancer cells, which areknown to be able to function as damage-associated molecularpatterns (DAMP) that induce host antitumor response (32). Wetherefore investigated the ability of RT53 treated tumor cells toactivate the adaptive immune system using a well-established

RT53 RT53M

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Figure 4.

Presence of RT53 at the plasma membrane of cancer cells. A, C8161 or HaCat cells were exposed to rhodamine-labeled RT53 or RT53M for 6 hours. Cells were thenfixed, stained for DNA, and examined by fluorescence microscopy. B and C, structural analysis of RT53. B, PEP-FOLD structural prediction of RT53. The sequencescorresponding to the penetratin moiety and the heptad leucine repeat of AAC-11 are in cyan and magenta, respectively. The experimental structure of the segmentcorresponding to the penetratin moiety of RT53 is shown in the left inset, and the experimental structure of AAC-11 is shown in the right inset with the helixcorresponding to the heptad leucine repeat in magenta. The two leucine residues that are mutated in glycines in RT53M are shown in orange. C, predictedtransmembrane orientation of RT53. In both predicted orientations, RT53 is displayed as a solid surface, and the spheres crudely represent amembrane surface. Thetwo leucine residues that are mutated in glycine in RT53M are shown in orange.

Characterization of an AAC-11–Derived Anticancer Peptide

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vaccination assay in immunocompetent C57BL/6 mice (33).In vitro experiments demonstrated that RT53 efficiently killedB16F10 murine melanoma cells (Fig. 7A) and, as described forhuman melanoma cell lines, RT53-treated B16F10 cellsreleased both HMGB1 and ATP (not shown). Very interestingly,vaccination of C57BL/6 mice with RT53-killed B16F10 cellsinduced tumor growth inhibition at the challenge site com-pared with control mice (Fig. 7B), strongly suggesting thatRT53-treated cells can activate the adaptive immune system.Notably, tumor growth was absent in 25% of the animals 30days after challenge with live cells, while all the control animalsdeveloped tumors at the challenge site within 7 days afterinoculation (Fig. 7C). Combined, these data indicate that RT53elicits immunogenic cell death and that RT53 treatment ofB16F10 melanoma cells mediates anticancer effect in a pro-phylactic tumor vaccination model.

DiscussionIn this study, we conducted proof-of-principle experiments for

testing the therapeutic value of using RT53 as a novel cancertreatment agent. RT53 selectively killed multiple cancer cell linesin vitro, while sparing nonmalignant cells, through membrano-lysis, leading to release of DAMPs. Interestingly, RT53 can inhibittumor growth in vivo in xenotransplant melanoma models, BRAFwild-type and mutant, without off-target toxicity, and RT53-

treated mouse melanoma cells mediated anticancer effects in aprophylactic tumor vaccination model.

RT53 inserts in the membrane of cancer cells, but not normalcells, suggesting binding with a membrane partners that is absentor minimally present in the membranes of untransformed cells.This binding could allow the peptide to accumulate in the cancercells' membranes where it could undergo pore formation andinduce necrosis, as described for otherMAPs (34). The inability ofRT53 to alter normal cells as well as mitochondrial membranesfurther supports the idea that RT53 cytotoxicity toward cancercells is not due to an unspecific detergent-like effect on the plasmamembrane. Therefore, our hypothesis is that the cytotoxic effect ofRT53 results from a two-step mechanism: first RT53 binds,through its AAC-11 sequence, to cancer cell–specific targets onthe cell membrane. Second, after a critical threshold concentra-tionof thepeptide is reached, amarkedmembranedepolarizationoccurs, caused by pore formation, leading to cell death. This is inline with our observations that RT53 induces necrotic tumor celldeath only when used at a certain concentration. Interestingly, anRT53 proposedmechanism of action is very reminiscent to that ofanother anticancer, MAP called PNC-27. PNC-27, which containsan HDM-2-binding domain derived from p53 fused to the pene-tratin sequence, induces membrane leakage of cancer cells, butnot normal cells, through binding to HDM-2 in the transformedcell plasma membranes (35–37). Structure prediction analysissuggests that like PNC-27 (37), RT53possesses amembrane active

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RT53 inhibits melanoma tumor growth in vivo as a single agent. A, RT53 in vivo toxicity. Groups of female nude mice were exposed to either single (left) or for 5consecutive days (gray; right) i.p. administrations of increasing doses of RT53. B, male FVB/N mice were immunized with 125 mg of RT53 daily for 5 weeks.Immunoglobulin level in blood samples was detected using an ELISA. C and D, effect of RT53 in C8161 (C) and SK-Mel-28 (D) melanoma xenograft models. Animalswere treated with i.p. injections of RT53 or RT53M in normal saline at daily doses of 5 mg/kg or normal saline as control.

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conformation. At this moment, the identity of RT53 membranepartner(s) is still unknown. RT53 is still cytotoxic against cancercells following AAC-11 knockdown. Even known, we cannot ruleout anoff-target effect of thepeptide that could result in cancer celldeath in the absence of AAC-11, our data strongly suggest thatAAC-11 is not a membrane partner of RT53. Preliminary experi-ments indicate that among the currently known AAC-11 inter-actors, FGF2, Acinus, ALC1 (11, 15, 16), and ALC1 do not appearto be involved in RT53 retention in cancer cells' membrane(not shown). AAC-11, which is overexpressed in most cancercells, upregulates FGF2 signaling (13). As FGF2 is known to bepresent at the plasma membrane (38), we are investigatingwhether RT53 might bind to FGF2, which could mediate RT53membrane retention. Interestingly, FGF2 stimulation is knownto induce plasmamembrane translocation of the scaffold proteinc-Jun-NH2-terminal kinase (JNK)⁄stress-activated protein kinase-associated protein-1 (JSAP1; ref. 39). It is therefore possible thatAAC-11-mediated sustained FGF-2 signaling in cancer cells mightinduce membrane relocalization of RT53-binding proteins. Weare currently using genome-scale knockout screenings as well aspeptide pulldown-based strategies to gain insight into the identity

of the molecules that mediates RT53 membrane retention incancer cells.

Necrosis induces intracellular potassium effluxes. Potassiumefflux has been shown to play an important role in cell deathand caspase activation, and depletion of potassium withdepolarizing drugs was shown to cause caspase activation(40–42). Therefore, caspase activation by RT53 probably repre-sents a side effect caused by its pore-forming properties, aswitnessed with Staphylococcus aureus a-toxin, which induces celldeath in a necrotic-like manner, through insertion into theplasma membrane and subsequent pore formation, despitecaspase activation (43). This explains why caspase inhibitionusing a pan-caspase inhibitor did not prevent RT53-mediatedcell death.

Importantly, RT53 anticancer effects in vitro translated well ins.c. mouse xenografts with melanoma cells. Indeed, RT53 alonesubstantially reduced tumor growth of both BRAF wild-typeand mutant models, when injected systemically. Histologicalanalysis of RT53-treated tumors indicated both increasedtumor cell apoptosis and necrotic cytotoxicity, comparedwith controls. AAC-11 deregulation is known to make cancer

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Mechanistic basis for RT53-mediated tumor inhibition.A, ex vivo detection of rhodamine-labeled RT53 in organs and tumors. The spectrumgradient bar correspondsto the fluorescence intensity unit p/s/cm2. B, representative pictures of histological analysis of tumors treated with RT53, RT53M, or normal saline. Top,morphological details to assess necrotic areas were investigated using H&E staining. Middle, proliferation index was assessed by staining with anti-Ki-67 antibodies.The Ki-67 staining data are presented as a percentage of Ki-67–positive cells treated over control tumors. Bottom, apoptotic cells were detected by TUNEL staining.Nuclei were stained with DAPI. Apoptosis was quantified as a percentage of TUNELþ nuclei relative to the total nuclei.

Characterization of an AAC-11–Derived Anticancer Peptide

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cells more vulnerable to additional stress, such as nutritionalstress (12) and, when used at sub-cytotoxic doses, RT53decreases the prosurvival functions of AAC-11 and sensitizecancer cell to various factors in vitro, such as chemotherapeuticdrugs and serum deprivation (11). It is therefore likely thatRT53 makes cancer cells more vulnerable to environmentalfactors within the tumor, such as decreased oxygen tension, pH,and nutrient availability, hence increasing apoptosis in tumorsfrom RT53-treated mice. Based on these observations, wehypothesize that RT53 could behave as a chemosensitizer inclinical settings, hence potentiating the efficacy of chemother-apeutic agents, and we are now evaluating this hypothesis.Cancer cells treated with RT53 in vitro released DAMPs, suchas HMGB1 and ATP, suggesting that RT53-induced cell death isimmunogenic. Interestingly, RT53-killed B16F10 cells mediat-ed anticancer effect in syngeneic C57BL/6 mice in a tumorvaccination assay, indicating potent immune response in vivo.Experiments are currently under way to evaluate cross-primingand cytokine release of cytotoxic T lymphocytes induced byRT53-treated cells, to further examine the adaptive immuneresponse to the antitumor vaccination achieved with RT53-treated cells.

RT53 possesses favorable drug-like properties. It is not toxic,even at doses 20-fold higher than the efficacious dose, notimmunogenic, exhibits efficient biological effect and it reachesdistant tissues and organs, including subcutaneous tumors where

it accumulates. Therefore, our results demonstrate that peptide-based targeting of AAC-11 can constitute a promising newapproach in the treatment ofmelanoma and possibly a significantfraction of human cancers.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: L. Jagot-Lacoussiere, B.O. Villoutreix, H. Bruzzoni-Giovanelli, J.-L. PoyetDevelopment of methodology: L. Jagot-Lacoussiere, E. Kotula, J.-L. PoyetAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): L. Jagot-Lacoussiere, E. Kotula, B.O. VilloutreixAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): L. Jagot-Lacoussiere, E. Kotula, B.O. Villoutreix,H. Bruzzoni-Giovanelli, J.-L. PoyetWriting, review, and/or revision of the manuscript: L. Jagot-Lacoussiere,B.O. Villoutreix, H. Bruzzoni-Giovanelli, J.-L. PoyetAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): E. Kotula, J.-L. PoyetStudy supervision: J.-L. Poyet

AcknowledgmentsWe thank Prof.Nicole Basset-Seguin andDrs.MarieDutreix, Robin Fa

�hraeus,

and Nicolas Dumaz for providing cell lines. We gratefully thank the coworkersof the Animal Experimental Facilities and the Imagery Department of the IUH.

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Figure 7.RT-53 treatment induces tumor protection from B16F10 cells. A, viabilityof B16F10 cells exposed to increasing concentrations of RT53 for20 hours. B, tumors growth on the challenge site of mice used in theprophylactic tumor vaccination experiments. The statistical differencefrom control is shown in the vaccination group. C, evolution of tumorincidence over time as Kaplan–Meier curve.

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Grant SupportThis work was supported by the INSERM and a grant from INSERM Transfert

and SATT IDF Innov.The costs of publication of this article were defrayed in part by the

payment of page charges. This article must therefore be hereby marked

advertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received February 2, 2016; revised June 22, 2016; accepted June 24, 2016;published OnlineFirst July 12, 2016.

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2016;76:5479-5490. Published OnlineFirst July 12, 2016.Cancer Res   Léonard Jagot-Lacoussiere, Ewa Kotula, Bruno O. Villoutreix, et al.   Cancer Cells DeathA Cell-Penetrating Peptide Targeting AAC-11 Specifically Induces

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