The New Antitumor Drug ABTL0812 Inhibits the Akt/mTORC1 … · Cancer Therapy: Preclinical The New Antitumor Drug ABTL0812 Inhibits the Akt/mTORC1 Axis by Upregulating Tribbles-3

Cancer Therapy: Preclinical

The New Antitumor Drug ABTL0812 Inhibits theAkt/mTORC1 Axis by Upregulating Tribbles-3PseudokinaseTatiana Erazo1, Mar Lorente2,3, Anna L�opez-Plana4, Pau Mu~noz-Guardiola1,5,Patricia Fern�andez-Nogueira4, Jos�e A. García-Martínez5, Paloma Bragado4,Gemma Fuster4, María Salazar2, Jordi Espadaler5, Javier Hern�andez-Losa6,Jose Ramon Bayascas7, Marc Cortal5, Laura Vidal8, Pedro Gasc�on4,8,Mariana G�omez-Ferreria5, Jos�e Alf�on5, Guillermo Velasco2,3, Carles Dom�enech5, andJose M. Lizcano1

Abstract

Purpose: ABTL0812 is a novel first-in-class, small moleculewhich showed antiproliferative effect on tumor cells in pheno-typic assays. Here we describe the mechanism of action of thisantitumor drug, which is currently in clinical development.

Experimental Design:We investigated the effect of ABTL0812on cancer cell death, proliferation, and modulation of intracel-lular signaling pathways, using human lung (A549) and pancre-atic (MiaPaCa-2) cancer cells and tumor xenografts. To identifycellular targets, we performed in silico high-throughput screeningcomparing ABTL0812 chemical structure against ChEMBL15database.

Results: ABTL0812 inhibited Akt/mTORC1 axis, resulting inimpaired cancer cell proliferation and autophagy-mediated celldeath. In silico screening led us to identify PPARs, PPARa andPPARg as the cellular targets of ABTL0812. We showed thatABTL0812 activates both PPAR receptors, resulting in upregula-

tion of Tribbles-3 pseudokinase (TRIB3) gene expression. Upre-gulated TRIB3 binds cellular Akt, preventing its activation byupstream kinases, resulting in Akt inhibition and suppression ofthe Akt/mTORC1 axis. Pharmacologic inhibition of PPARa/g orTRIB3 silencing prevented ABTL0812-induced cell death.ABTL0812 treatment inducedAkt inhibition in cancer cells, tumorxenografts, and peripheral bloodmononuclear cells frompatientsenrolled in phase I/Ib first-in-human clinical trial.

Conclusions:ABTL0812has a unique and novelmechanismofaction, that defines a new and drugable cellular route that linksPPARs to Akt/mTORC1 axis, where TRIB3 pseudokinase plays acentral role. Activation of this route (PPARa/g-TRIB3-Akt-mTORC1) leads to autophagy-mediated cancer cell death. Giventhe low toxicity and high tolerability of ABTL0812, our resultssupport further development of ABTL0812 as a promising anti-cancer therapy. Clin Cancer Res; 22(10); 2508–19. �2015 AACR.

IntroductionThe PI3K/AKT/mTORC1 (mTOR complex-1) signaling path-

way drives signals from ligand-stimulated receptor tyrosinekinases (RTK) to proteins that control cell metabolism, growth,size, survival, and angiogenesis (1). In response to growth orsurvival factors, phosphorylated RTKs activate PI3K, which gen-erates the phosphatidylinositol 3,4,5-trisphosphate (PIP3) sec-ondmessenger and allows the recruitment of protein kinaseAkt tothe cellular membrane. Akt binds PIP3 through its PH domain,resulting in a conformational change that enables its activationthrough phosphorylation of two critical residues: Thr308 at theT-loop of the kinase domain by the phosphoinositide-dependentkinase-1 (PDK1), and Ser473 at the C-terminal hydrophobicmotif by mTOR complex-2 (mTORC2; refs. 2, 3). Active Aktphosphorylates a plethora of substrates that regulate cell survivaland metabolism, and also cell growth through subsequent acti-vation of mTORC1. Akt phosphorylation of PRAS40 and TSC2leads to activation of mTORC1, which promotes protein trans-lation and synthesis by phosphorylating ribosomal S6-Kinase(S6K) and the elongation factor 4EBP-1 (4).

Human cancer is very often associated with activation of thePI3K/Akt/mTORC1 pathway, mainly due to overexpression or

1Protein Kinases and Signal Transduction Laboratory, Institut de Neu-roci�encies and Departament de Bioquímica i Biologia Molecular, Uni-versitat Aut�onoma de Barcelona, Bellaterra, Barcelona, Spain.2Department of Biochemistry and Molecular Biology, Faculty ofBiology, Universidad Complutense, Madrid, Spain. 3Instituto deInvestigaci�on Sanitaria del Hospital Clínico San Carlos (IdISSC),Madrid, Spain. 4Area of Molecular and Translational Oncology,IDIBAPS, Fundaci�o Clínic, Universitat de Barcelona, Barcelona, Spain.5Ability Pharmaceuticals, SL, Edifici Eureka, Campus UAB, Bellaterra,Barcelona, Catalonia, Spain. 6Pathology Department, Hospital Uni-versitari Vall d'Hebron, Barcelona, Spain. 7Institut de Neuroci�enciesand Department of Biochemistry and Molecular Biology, UniversitatAut�onoma de Barcelona, Barcelona, Spain. 8Medical OncologyDepartment, Novel Therapeutics Unit, Hospital Clínic Barcelona.Barcelona, Catalonia, Spain.

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

Corresponding Author: Jose M. Lizcano, Institut de Neuroci�encies and Departa-ment de Bioquímica i Biologia Molecular, Facultat de Medicina, UniversitatAut�onoma de Barcelona, Bellaterra, Barcelona 08193, Spain. Phone: 34 935-813-076; Fax: 34 935-811-573; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-15-1808

�2015 American Association for Cancer Research.

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activatingmutations of RTKs and PI3K, to deletions/mutations ofthe PIP3 phosphatase PTEN and to amplification of Akt (3, 5).These types of tumors are hypersensitive to inhibition of each ofthe components of the PI3K/Akt/mTORC1 axis, and thereforemajor efforts have been taken to develop inhibitors of thissignaling pathway (6). Akt acts as a central node of this pathway,and hyperactivated Akt is a common feature observed in humansolid tumors (3, 5). Therefore, Akt has been proposed as aninteresting target in cancer therapy. Two classes of Akt inhibitorsare now in clinical development: ATP-competitive inhibitors(such as GSK690693) which target active Akt, and allostericinhibitors (such as MK-2206 and perifosine, currently in phaseII and phase III clinical trials, respectively) that by binding the PHdomain of Akt prevent its activation by upstream kinases (3, 7).

Here we present ABTL0812, a first-in-class molecule with anti-tumor activity which is currently in phase I/Ib clinical trials inpatients with advanced solid tumors. We provide evidences show-ing that ABTL0812 inhibits Akt phosphorylation by upregulatingthe expressionofTRIB3,apseudokinase thatbindsand inhibitsAkt,leading tomTORC1 inhibitionandautophagy-mediated cell death.

MethodsCell culture, viability assay, transfection, and lysis

Human cancer cell lines were purchased from ATCC (2008;ATCC-authentication by isoenzymes analysis). Atg5þ/þ andAtg5�/� T-large antigen–transformed MEFs were donated by Dr.Mizushima (Tokyo Medical University, Tokyo, Japan). TRB3þ/þ

and TRB3�/� RasV12/E1A–transformed Mouse embryonic fibro-blasts (MEF) have been described before (8). Cell lines werecultured as recommended and transfected using Lipofectamine-2000.Cell viabilitywas determinedby theMTT (3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium Blue) reduction assay(9). Cells were lysed in ice-cold RIPA buffer supplemented with 1mmol/L sodium-orthovanadate, 50 mmol/L NaF, and 5 mmol/Lsodium-pyrophosphate, sonicated and stored at �20�C.

Immunoblotting and immunoprecipitationImmunoblot analyses were performed following standard

procedures as described (10) and antibodies listed in the Sup-

plementary Table S1. Akt was immunoprecipitated following theprotocol previously described (11).

Transmission electron microscopyCells treated with ABTL0812 or vehicle (ethanol) were pro-

cessed as described before (12). Pellets were embedded in Epo-nate-12TM resin (Ted Pella). Ultrathin (70-nm thick) sectionswere contrastedwith uranyl-acetate and lead citrate solutions, andvisualized in transmission electron microscope (Jeol JEM-1400)equipped with a CCD- GATAN-ES1000W Erlangshen camera.

Xenograft modelsFor A549 model, athymic female nude mice (n ¼ 5 per group)

were injected subcutaneously with 5 � 106 cells in each flank.When tumors reached 80 to 100 mm3, mice were randomlydistributed into treatment groups and administered the corre-sponding treatments. ABTL0812 was administered by oral gavageat 120 mg/kg every day. Docetaxel was administered by intraper-itoneal route at 15 mg/kg once a week. MiaPaCa-2 cells wereinjected in athymic male mice (n ¼ 5 per group) with 10 � 106cells in one flank. When tumors reached 80 to 160 mm3, micewere randomly distributed and treated with vehicle or ABTL0812120 mg/kg by oral gavage five times per week. Tumor volumeswere measured as (length � width2)/2, twice a week. All proce-dures involving animal were performed with the approval of theHospital Clinic Animal Experimentation Committee, accordingto Spanish official regulations.

Pharmacokinetics of ABTL0812 in mice and ratsABTL0812 was orally (gavage, 100 mg/kg) and intravenously

(10 mg/kg) administered to male CD-1 mice. ABTL0812 wasquantified in plasma by validated LC/MS-MS methods and phar-macokinetic parameters were calculated with WinNonlinsoftware.

Clinical trial and isolation of PBMC from patientsABTL0812 is currently in clinical evaluation in a phase I/Ib trial

in patients with advanced solid tumors. This study was approvedby the local Ethics Committee of the Hospital Clinic Barcelonaand the Spanish Agency of Medicines. The trial is registered atwww.clinicaltrials.gov (NCT02201823). ABTL0812 was admin-istered daily by the oral route at a dose of 1,000mg twice daily fora period of 28 days. Blood samples for biomarker study wereobtained by venipuncture before and after ABTL0812 treatment.PBMC were isolated by Ficoll (GE-Healthcare) density centrifu-gation, lysed in RIPA buffer, sonicated, and stored at �20�C.

Statistical analysisAll in vitro data were assessed using one-way ANOVA followed

by Bonferroni multiple comparison test. Tumor volumes of micewere compared using the ANOVA followed by t test. Statisticalsignificance between the groups was assessed with the log-ranktest (GraphPad). Levels of statistical significance were set atP � 0.05.

ResultsABTL0812 has anticancer activity in vitro and in vivo

A novel class of chemically modified lipid-derived small mole-cules was selected on the basis of two phenotypic assays: anti-proliferative effect on tumor cells and low toxicity after high doses

Translational Relevance

Hyperactivation of the Akt/mTORC1 axis is commonlyobserved in many human cancers and blocking this pathwayis an important anticancer strategy. Inhibition of Akt is a validtarget for anticancer therapy and several AKT inhibitors areunder clinical development. We present ABTL0812, a first-in-class small molecule with a unique mechanism of action. Incells and tumor xenografts models, ABTL0812 induces upre-gulation of TRIB3 pseudokinase. TRIB3 binds Akt, preventingits activation by upstream kinases and resulting in an effectiveinhibition of the Akt/mTORC1 axis and in autophagy-medi-ated cancer cell death. ABTL0812 is currently in phase I/Ib first-in-human clinical trial in patients with advance solid tumors.Preliminary results show that ABTL0812 also induces Aktinhibition in humans. These evidences, together with a verylow toxicity and high tolerability, support further develop-ment of ABTL0812.

Inhibition of Akt/mTORC1 Axis by TRIB3 Pseudokinase

www.aacrjournals.org Clin Cancer Res; 22(10) May 15, 2016 2509




Figure 1.ABTL0812 induces cancer cell death in vitro and reduces tumor growth in human lung andpancreatic xenografts. A, chemical structure of ABTL0812. B, ABTL0812 cellviability assays in human cancer cell lines. IC50 values were obtained in MTT assays after exposure to ABTL0812 for 48 hours. Values are the mean � SDof three separate determinations. (Continued on the following page.)

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Clin Cancer Res; 22(10) May 15, 2016 Clinical Cancer Research2510




administration in rodents. From this class, ABTL0812was selectedfor further preclinical development. The formula for ABTL0812 is2-hydroxylinoleic acid (Fig. 1A).

ABTL0812 reduced viability of different human cancer cell linestestedwith IC50 values of 20 to 60 mmol/L (Fig. 1B). Real-time cellanalyses further demonstrated the antiproliferative effect ofABTL0812 (Supplementary Fig. S1). ABTL0812 also induced celldeath, which was confirmed by propidium iodide staining (Sup-plementary Fig. S2). Importantly, ABTL0812 induced death incancer cells but not in normal cells. Compared with LN-18glioblastoma cells, primary astrocytes were resistant to ABTL0812treatment at the highest ABTL0812 concentration tested (200mmol/L; Fig. 1C).

To address ABTL0812 pharmacokinetics, we administered thecompound to mice by oral and intravenous routes. ABTL0812showed an excellent bioavailability by oral route (F:103%),with rapid oral absorption (tmax:30 minutes), high pick concen-trations (Cmax:2.0 mg/mL), and wide volume of distribution(Vss:10.4 L/kg). Clearance was also high (Cl: 82 mL/minute/kg).Overall, the compound was well tolerated and animals appearedhealthy with no clinical signs of distress, local or systemic toxicity.

To test the efficacy of ABTL0812 in inhibiting tumor growth invivo, we used human lung and pancreatic xenograft models. A549human lung cancer xenografts of nude mice were treated withABTL0812 or the standard-of-care docetaxel. ABTL0812 inhibitedtumor progression with an efficacy similar to docetaxel (Fig. 1D).Moreover, ABTL0812 lacked the toxic effects observed for doc-etaxel, as shown by body weight measurements. Similarly,ABTL0812 also inhibited the growth of MiaPaCa-2 human pan-creatic cancer xenografts in nude mice, without affecting bodyweight (Fig. 1E).

ABTL0812 induces autophagy-mediated cell deathNext, we investigated the type of cell death induced by

ABTL0812 in A549 and MiaPaCa-2 cells. ABTL0812 did notinduce nuclear fragmentation/condensation, or caspase-3 activa-tion (Supplementary Fig. S3). Treatment with staurosporineresulted in typical features of apoptosis, showing that A549 andMiaPaCa-2 cells are apoptosis competent. These results indicatethat ABTL0812 fails to induce apoptosis. In turn, several evidencesindicated that ABTL0812-induced cell death is mediated byautophagy.

Two of the hallmarks of autophagy are the conversion of thesoluble form of LC3 to a lipidated form associated to autophago-somes (LC3-II), and the elimination of autophagosome cargoproteins such as p62 (13). In A549 and MiaPaCa-2 cells,ABTL0812 induced the appearance of LC3-II and a decrease onp62 protein in a concentration-dependent manner (Fig. 2A). Wealso observed the occurrence of LC3-positive dots in cells treatedwith ABTL0812, which indicates association to autophagosomes(Supplementary Fig. S4). Electron microscopy analysis ofABTL0812-treated A549 and MiaPaCa-2 cells revealed the pres-ence of double membrane vacuolar structures with the morpho-logic features of autophagosomes (Fig. 2B). Moreover, ABTL0812

also induced hallmarks of autophagy in vivo, as tumors fromA549xenograftmice treatedwithABTL0812 showed an increase in LC3-II levels, compared with mice treated with vehicle (Fig. 2C).

Next, we investigated the role of autophagy in the cell deathinduced by ABTL0812. Pharmacologic inhibition of autophagywas achieved by treating cells with a combination of the lyso-somal protease inhibitors E64d and Pepstatin-A (PA) that blockthe final step of autolysosomal degradation. Treatment of cellswith E64dþPA prevented ABTL0812-induced cell death andresulted in an enhancement of LC3-II accumulation in cellstreated with ABTL0812 (Fig. 2D), indicating that ABTL0812induces dynamic autophagy in cancer cells. Finally, we evaluatedthe activity of ABTL0812 in oncogene-transformed embryonicfibroblasts derived from Atg5�/� (autophagy-deficient) mice.Atg5 is an essential protein for autophagosome formation(14). Transformed Atg5�/� MEF cells were more resistant toABTL0812-induced cell death than transformed Atg5þ/þ MEFcells, and did not activate autophagy in response to ABTL0812treatment (Fig. 2E). Taken together, these results show thatautophagy mediates ABTL0812-induced cancer cell death.

ABTL0812 inhibits the Akt/mTOR axisAutophagy induction is often consequential to the inhibition

of mTOR signaling, as mTORC1 acts as a central regulator inautophagy induction (15). We therefore investigated whetherABTL0812-induced autophagy and cancer cell death occurred viamTORC1 inhibition, by measuring levels of activation of com-ponents of the Akt/mTORC1 signaling pathway. A549 and Mia-PaCa-2 cells showed measurable phosphorylation levels for allthe proteins of Akt/mTORC1 axis in basal conditions. ABTL0812treatment reduced phosphorylation of ribosomal-S6 kinase andof ribosomal-S6 protein (a well-established S6K substrate) in aconcentration-dependent manner (Fig. 3).

Protein kinase Akt modulates the activation of mTORC1 byphosphorylating and inactivating the TSC2 and PRAS40 proteinsthat act as repressors ofmTORC1 activity (1). Thus, Akt inhibitionresults in impairedmTORC1 activity and activation of autophagy.ABTL0812 induced Akt inhibition in A549 andMiaPaCa-2 cells ina dose-dependent manner, by reducing phosphorylation of thetwo critical residues involved in Akt activation, Thr308 andSer473. Consequently, ABTL0812 also reduced phosphorylationof theAkt substrates PRAS40 andTSC2 (Fig. 3). These results showthat ABTL0812 induced inhibition of the Akt/mTORC1 axis incancer cells.

ABTL0812 binds and induces PPARa and PPARgtranscriptional activities, which mediate in ABTL0812-inducedcancer cell death

To identify themolecular targets of ABTL0812, we performed insilico high-throughput screening comparing ABTL0812 chemicalstructure against the ChEMBL15 database (https://www.ebi.ac.uk/chembldb/), following the similar property and active analogprinciples (16). This approach allowed the identification ofseveral ABTL0812 target candidates, including PPARa andPPARg .

(Continued.) C, LN-18 glioblastoma cells or primary rat astrocytes were incubated with ABTL0812, and 48 hours later cell viability was monitored by MTTassays. Similar results were obtained in three separate determinations. D and E, ABTL0812 inhibits tumor growth in lung (D) and pancreatic (E) cancer xenograftmodels. Athymic nude mice injected with A549 or MiaPaCa-2 cells were treated with vehicle or ABTL0812 by oral gavage. Docetaxel was administered byintraperitoneal injection. Tumor growth and weight variation curves are shown. Results are the mean � SD of five mice in each group. Because of toxicityin the docetaxel treatment, only two mice were left at day 25 (n ¼ 2) and one after day 28 (n ¼ 1). � , P < 0.05; �� , P < 0.01.






Figure 2.ABTL0812 induces autophagy in vitro and in vivo. Autophagy mediates ABTL0812-induced cancer cell death. A, cells were treated with 50 to 200 mmol/LABTL0812 for 12 hours, lysed and levels of the autophagy-marker proteins LC3 and p62 visualized by immunoblotting. B, representative electron microscopymicrophotographs showing autophagosomes (right) in cells treated with 50 mmol/L ABTL0812 for 10 hours. C, total protein extracted from A549 xenografttumors (Fig. 1C) were analyzed by immunoblotting for expression of LC3. Figure shows a representative analysis from vehicle (n ¼ 4) and treated (n ¼ 5)tumors. LC3-II levels were normalized with actin and estimated in densitometric units. D, A549 (white columns) or MiaPaCa-2 (black columns) cells werepreincubated with vehicle or a combination of lysosomal protease inhibitors E64d (10 mmol/L) and Pepstatin-A (PA, 10 mg/mL), before treatment with50 mmol/L ABTL0812 for 24 hours. Cell viability was determined by MTT assay. Each value is the mean � SD of three different experiments. ��P < 0.001 fromABTL0812 treated cells. Cells were lysed and LC3 lipidationwas visualized by immunoblotting. E, Atg5þ/þ and Atg5�/�MEFswere treatedwith vehicle or ABTL0812and cell viability determined 24 hours later. Values are the mean � SD of three different experiments. � , P < 0.05; �� , P < 0.001 from Atg5þ/þ MEFs.

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Of note, a possible interaction with PPARb/d was not found.Putative ABTL0812-bindingmolecules identified in this screeningwere further investigated through docking analysis. Figure 4Ashows themolecular model of ABTL0812 into the ligand-bindingpocket of PPARg . In this model, ABTL0812 may bind the PPARgligand-binding domain (LBD) through its carboxylate group,which forms hydrogen bonds with the side of key amino acidresidues (Ser289, His323, Tyr473, and His449), as it has beenshown for other PPARg agonists such as rosiglitazone (17). The2-hydroxyl group of ABTL0812 may also form hydrogen bondswith residues Cys285 and Ser289, which are also conserved in thePPARa LBD. This molecular model proposes that ABTL0812might establish interactions similar to the specific PPARg agonistrosiglitazone, thus predicting that ABTL0812 could act as PPARagonist. Indeed, in vitro radioligand displacement assays usingpurifiedproteins showed that ABTL0812binds PPARa andPPARgligand-binding pockets with Ki values of 7.1 mmol/L and 4.7mmol/L, respectively (Supplementary Fig. S5).

Next, we investigated the functional relevance of this interac-tion, performing luciferase-based gene reporter assays in cellstransfected with a PPAR-response element (PPRE) fused to lucif-erase construct. In A549 and MiaPaCa-2, ABTL0812 inducedapproximately 5-fold PPAR transcriptional activity, similarly tothe specific agonistsWY-14643 (PPARa) or rosiglitazone (PPARg ;Supplementary Fig. S6). To determine whether ABTL0812-induced PPAR activation was mediated by the PPAR isoformsa and/or g , we performed analogous gene reporter assays in cellstransiently transfected with plasmids encoding each isoform.ABTL0812-induced PPAR transcriptional activity was higher incells overexpressing PPARa or PPARg proteins, compared withthose having the endogenous proteins only (Fig. 4B), suggestingthat ABTL0812 functionally interacts with both receptors in cells.

Finally, we performed cell viability assays in the presence ofPPARa/g antagonists. In A549 and MiaPaCa-2 cells, preincuba-tion with PPARa antagonist GW6471 or PPARg antagonistGW9662 prevented cell death induced by ABTL0812 (Fig. 4C),indicating that PPAR receptorsmediate in ABTL0812-induced celldeath.

ABTL0812 activates PPARa- and PPARg-dependent expressionof TRIB3

Koo and colleagues showed that PPARs regulate the expressionof the pseudokinase Tribbles 3 (TRIB3), through binding a PPAR-responsive element sequence within the TRIB3 promoter (18).Other authors have shown that TRIB3 can interact with Akt,preventing its activation by upstream kinases and resulting in aninactive form of Akt (8, 19). Thus, we hypothesized thatABTL0812 could inhibit Akt (and downstream mTORC1) byupregulating TRIB3 via PPARa/g activation. In A549 and Mia-PaCa-2 cells, ABTL0812 induced a quick and robust increase inTRIB3 protein levels after 12 hours of treatment (Fig. 5A), thatcorrelated with an increase in gene transcription (Fig. 5B) andqPCR-measured TRIB3 mRNA levels (Fig. 5C). Although wecannot discard an effect of ABTL0812 on TRIB3 stability, theseresults indicate that ABTL0812 upregulates TRIB3 gene transcrip-tion. Furthermore, treatment with PPARa and/or PPARg inhibi-tors GW6471 and GW9662, respectively, completely abolishedthe ABTL0812-induced upregulation of TRIB3 gene expression(Fig. 5C). Interestingly, treatment with either PPARa or PPARgagonists resulted in increased TRIB3 expression and reduced Aktphosphorylation (Fig. 5D). However, it was necessary to combinetreatment with both agonists to obtain similar levels of inhibitionof Akt (phospho-PRAS40) andmTORC1 activities (phospho-S6Kand phospho-S6) than those induced by ABTL0812. Altogether,our results demonstrate that ABTL0812 induces TRIB3 geneexpression via activation of PPARa and PPARg activities.

ABTL0812 inhibits Akt and mTORC1 via TRIB3To investigate the role of upregulated TRIB3 on Akt activation,

we overexpressed increasing amounts of TRIB3 in A549 andMiaPaCa-2 cells. We observed reduced phosphorylation of Aktand Akt substrate PRAS40 that inversely correlated with TRIB3expression (Fig. 6A). This was probably due to a TRIB3–Aktphysical interaction, because ABTL0812 treatment increased theamount of Akt coimmunoprecipitated with TRIB3 (Fig. 6B).Moreover, unlike wild-type transformed MEF, TRIB3-deficientMEF cells did not show Akt (Ser473) or mTORC1 (pS6) inhibi-tion, or induction of autophagy (LC3-II marker) in response toABTL0812 treatment (Fig. 6C). Cell viability assays showed thatTRIB3-deficient MEFs are resistant to ABTL0812-induced celldeath, unlike wild-type transformedMEFs (Fig. 6D). These resultssupport a pivotal role of TRIB3 on the impairment of the Akt/mTORC1 axis and on the autophagy-mediated cancer cell deathobserved in response to ABTL0812 treatment.

ABTL0812 induces TRIB3 upregulation and Akt inhibitionin vivo

To determine the in vivo relevance of our findings, we nextinvestigated the effect of ABTL0812 in A549 human lung andMiaPaCa-2 human pancreatic cancer xenografted mice (Fig. 1Cand D). Immunohistochemical and immunoblotting analysesshowed that ABTL0812-induced upregulation of TRIB3 protein

Figure 3.ABTL0812 inhibits the Akt/mTORC1 axis. A549 and MiaPaCa-2 cells treatedwith vehicle (0) or ABTL0812 (mmol/L) for 24 hours were lysed, and levels ofphosphorylated proteins were analyzed by immunoblotting. Levels of totalproteins and actinwere used as control. Similar results were obtained in threeseparate experiments.






and inhibition of Akt phosphorylation (Ser473) in A549 andMiaPaCa-2 tumors (Fig. 6E). These data, together with that pre-sented in Fig. 2 showing that ABTL0812 induces autophagy inA549 xenografts, suggest that the antitumor activity of ABTL0812in vivo relies on inhibition of Akt and induction of autophagy-mediated cell death.

Akt phosphorylation is a pharmacodynamic marker in patientstreated with ABTL0812

Finally, we evaluated the activity of ABTL0812 in some patientsincluded in phase I/Ib clinical trials (NCT02201823), monitoringAkt phosphorylation levels in PBMCs. We collected blood sam-ples, pretreatment and at different days of chronic treatment with1,000 mg of ABTL0812, a dose that was safe and well tolerated(preliminary data not shown). Our preliminary results show that

treatment with ABTL0812 resulted in a marked reduction on Aktphosphorylation in PBMCs from 3 patients (Fig. 6F). We couldnot detect TRIB3 protein in PBMCs, using commercially availableanti-TRIB3 antibodies. However, parallel qPCR analysis showed asignificant increase in TRIB3 mRNA levels in human PBMCstreated in vitro with ABTL0812 (Supplementary Fig. S7), thussuggesting that ABTL0812 also induces Akt inhibition in humansthrough upregulation of TRIB3.

DiscussionHere,wepresent themechanismof actionofABTL0812, a novel

and first-in-class antitumor drug which is currently in phase I/Ibclinical trials in patientswith advanced solid tumors. ABTL0812 isa polyunsaturated fatty acid derivative, small molecule that

Figure 4.ABTL0812 induces PPARa and PPARg transcriptional activities, which mediate in ABTL0812-induced cancer cell death. A, model for the binding of ABTL0812(depicted in yellow) to PPARg ligand binding domain, showing the interactions (dotted green lines) of the carboxylate and hydroxyl groups of ABTL0812(depicted in red) with the side chains of residues (depicted in blue) within PPARg active site. B, luciferase-PPRE reporter and pRL-CMV-Renilla plasmidswere cotransfected with plasmids encoding empty backbone or PPARa or PPARg . Sixteen hours posttransfection, cells were treated with vehicle or50 mmol/L ABTL0812. Twenty-four hours later, lysates were subjected to dual-luciferase assay. Values are the mean � SD of three different experiments,each performed in triplicate and normalized using Renilla values. ��P < 0.001 from cells transfected with empty plasmid. C, cells were preincubated withvehicle (white columns), 0.2 mmol/L GW6471 (PPARa-antagonist) or 1 mmol/L GW9662 (PPARg-antagonist) during 2 hours, and then treated with50 mmol/L ABTL0812 for 24 hours. Cell viability was measured by MTT analysis. Values are the mean � SD of three different determinations. � , P < 0.05;�� , P < 0.001 from ABTL0812 treated cell.

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induces cell death in a broad panel of cancer cell lines, whereas itdoes not affect cell viability of nontumorogenic cells. ABTL0812also inhibits tumor growth in human lung and pancreatic tumorxenografts. Herein, we elucidated a novel cell signaling pathwaythrough which ABTL0812 induces autophagic cell death. Weshowed that ABTL0812 induces upregulation of the TRIB3 pseu-dokinase through activation of PPARa and PPARg transcriptionfactors.Upregulated TRIB3binds toAkt andprevents its activationby PDK1 and mTORC2 upstream kinases, resulting in inhibitionof the Akt/mTORC1 axis and, therefore, autophagy-mediated celldeath (Fig. 6G).

Autophagy is an essential process that consists of selectivedegradation of cellular components, assuring the maintenanceof cellular homeostasis via lysosomal degradation pathways (13).The role of autophagy in cancer is still far from clear, as it can act asa tumor suppressor and tumor promoter, depending on tumor

type, stage, and genetic context, as well as on the duration andstrengthof the triggering stimuli (15, 20).Our results demonstratethat ABTL0812 induces autophagy-mediated cancer cell death invitro and in vivo, without activating cellular apoptosis. Theseresults are in agreementwith those reporting that polyunsaturatedfatty acid and derivatives exert their antiproliferative actionthrough activation of autophagy (21–23).

The majority of current anticancer treatments activate apopto-sis, and resistance to chemotherapy is a major challenge in cancer(24). Autophagy-mediated cell death has emerged as an alterna-tive to kill cancer cells without inducing resistance to apoptosisinducer drugs (25). mTORC1 plays a central role in regulatingcellular autophagy, and mTORC1 inhibitors induce autophagy-mediated cell death in many systems, such as AZD8055 inhepatocellular carcinoma (26) or Ku0063794 and temsirolimusin renal carcinoma (27). On the other hand, mTORC1 activation

Figure 5.ABTL0812 induces upregulation of the Akt endogenous inhibitor TRIB3 through PPARa/g activation. A, cells treated with ABTL0812 for the indicated timeswere lysed, and levels of TRIB3 and actin analyzed by immunoblotting. B, cells were transfected with luciferase-TRIB3 promoter reporter and pRL-CMV-Renillaplasmids. Sixteen hours posttransfection, cells were treated with vehicle or 100 mmol/L ABTL0812. Twenty-four hours later, lysates were subjected todual-luciferase assay. Values are the mean � SD of three different experiments, each performed in triplicate and normalized using Renilla values.C, cells were preincubated with vehicle or PPARa antagonist GW6471 (0.2 mmol/L) and/or PPARg antagonist GW9662 (1 mmol/L) for 2 hours, and treatedfor 24 hours with vehicle or 50 mmol/L ABTL0812. TRIB3 mRNA levels were monitored by RT-qPCR. Values are the mean � SD of three different determinations,each performed in duplicate. ��P < 0.001 from vehicle-treated cells. D, A549 cells were treated with PPARa agonist WY14643 (1 mmol/L) and/or PPARg agonistrosiglitazone (1 mmol/L) for 24 hours, lysed and proteins analyzed by immunoblotting.






Figure 6.ABTL0812-evoked TRIB3 upregulation inhibits Akt activity in vitro and in vivo. A, cells transfected with empty or increasing amounts of a plasmid encodinghuman-TRIB3 were lysed and levels of proteins monitored by immunoblotting. B, immunoprecipitation of Akt from MiaPaCa-2 lysates after treatment withvehicle or ABTL0812 for 24 hours. Top, levels of Akt and TRIB3 in the immunoprecipitates. C, wild-type and TRIB3-KO MEF cells were treated with ABTL0812for 24 hours, and levels of the indicated proteins were analyzed by immunoblotting. D, wild-type and TRIB3-KO MEFs were treated with ABTL0812 for24 hours, and cell viability measured by MTT analysis. Values are the mean � SD of three different determinations. ��P < 0.001 from TRIB3 wt. E, A549 andMiaPaCa-2 tumors were collected after sacrifice, and TRIB3 was evaluated by immunohistochemical analysis (left; scale bars: 100 mm). Levels ofphospho-Akt(Ser473) were evaluated in total protein extracts, by immunoblotting (right). Histograms show the corresponding quantifications.� , P < 0.01; �� , P < 0.005; �� , P < 0.001 from vehicle-treated tumors. F, phospho-Akt and Akt levels in PBMCs from patients before (day 0) or after1,000 mg bid ABTL0812 oral chronic treatment. Protein extracts were analyzed by immunoblotting for phospho-Akt-Ser473 and total Akt. Upper graphsshow the quantification of levels of phospho-Akt normalized with Akt protein, estimated by densitometric analysis (day 1 ¼ 100%). G, a model showing themechanism of action of ABTL0812.

Erazo et al.





is frequently associated with resistance to antitumor drugs (6). AsABTL0812 is a potent inhibitor of the Akt/mTORC1 axis, itsadministration in combination with standard chemotherapeuticdrugs might prove effective in therapy-resistant or apoptosis-refractory tumor. This has been shown for the mTORC1 inhibitoreverolimus (RAD001), which sensitizes papillary thyroid cancercells to treatment with doxorubicin by an autophagy-mediatedmechanism (28).

Here we present in vitro and in vivo evidences showing thatABTL0812binds to and activates PPARa andPPARg transcriptionalactivities, and that pharmacologic inhibition of both receptorsprevents ABTL0812-induced cancer cell death, suggesting thatPPARa/g are the primary molecular targets of ABTL0812 in thecell. PPARs are nuclear receptors that heterodimerize with theretinoid-X receptors to modulate gene transcription. Mammaliancell expresses three PPAR isoforms, PPARa, PPARb/d, and PPARg(29). Using an in silico analysis, we identified PPARa and PPARg astargets of ABTL0812, but not PPARb/d. Several authors have shownthat polyunsaturated and derivative long fatty acids activate PPARstranscriptional activities (21, 30). Interestingly, the docking experi-ments performed in our study show that the a-hydroxyl group inABTL0812 forms two extra hydrogen bonds with the critical resi-dues Cys285/Ser289 in PPARg LBD (also conserved in the PPARaLBD), compared with the nonhydroxylated linoleic acid. Accord-ingly, ABTL0812 showed higher PPARg agonistic activity (Ki �5mmol/L) than linoleate (79 mmol/L) or the PPARg natural agonistsflavonoids such as kaempferol (30 mmol/L; ref. 30).

PPARs regulate the expression of many genes involved inglucose and lipid metabolism (29, 31). Interestingly, activationof PPARg reduces cell proliferation and invasion, and enhancesapoptosis in different cancer models such as breast and hepato-carcinoma (21, 32), whereas activation of PPARa inhibitstumor growth and angiogenesis in mouse models (33–35).Therefore, activators of PPARa or PPARg have been proposedas anticancer drugs, and specific synthetic PPARa or PPARgagonists are in clinical trials for treating cancer, such as the PPARgagonist efatutazone or the PPARa agonist fenofibrate, in patientswith liposarcoma (NTC02249949) or multiple myeloma(NCT01965834), respectively. ABTL0812, by activating PPARaand PPARg , might well synergize the anticancer activity of eachreceptor. Importantly, single treatment of the cancer cells used inour study with either PPARa or PPARg agonist induced partialinhibition of mTORC1 activity, whereas combined treatmentwith both agonists resulted in a robust mTORC1 inhibition,similar to that obtained for ABTL0812 treatment (Fig. 5D). Giventhe fact that anewgenerationof dual PPARa/g agonists has shownpromising anticancer activity (i.e., TZD18 inbreast cancer; ref. 36),it will be of interest to investigatewhether PPARa/gmechanismofaction also involves inhibition of the Akt/mTORC1 axis.

Our most striking result is the observation that upregulation ofTribbles pseudokinase-3, through activation of PPARa/g recep-tors, plays a crucial role in the anticancer activity of ABTL0812.TRIB3 is a highly conserved protein which lacks critical catalyticresidues for binding ATP and therefore lacks kinase activity (37).Together with TRIB1 and TRIB2 proteins, TRIB3 define the Trib-bles subfamily of pseudokinases, which was first described inDrosophila as regulators of cell proliferation andmigration duringdevelopment (38). As it happens for other pseudokinases, TRIB3exerts its function in the cell interactingwith several proteins, suchas the transcription factors SMAD3 (39) and ATF-4 (40), ormembers of the MAPK family (41). Of note, few authors have

shown that TRIB3 can interact with Akt, preventing its phosphor-ylation by upstream kinases and resulting in suppression of theinsulin pathway (16, 19, 42). Here we show that ABTL0812-evoked TRIB3 binds Akt, and that TRIB3 overexpression in cellsresulted inAkt inhibition.Moreover, we also found that treatmentwith ABTL0812 enhances the interaction between TRIB3 and Akt,in line with previous data obtained in glioma cells and oncogene-transformed MEFs, where a similar increase in TRIB3 levels andTRIB3-Akt interaction occur upon exposure to D9-tetrahydrocan-nabinol (THC; refs. 12). TRIB3 interacts more strongly withinactive/dephosphorylated Akt than active Akt (M. S., unpub-lished results), therefore it is likely that overexpressed TRIB3could trap the inactive form of Akt, preventing its activation/phosphorylation by upstream kinases. Whether TRIB3 masks thephosphorylatable residues in Akt (Thr308 and Ser473), induces aconformational change that impairs recognition by upstreamkinases PDK1 and mTORC2, or hampers the binding of the AktPHdomain to PIP3 remains to be clarified.On the other hand, it isalso likely that, in addition to changes in TRIB3 protein levels,there could exist additional mechanisms involved in regulatingthe interaction of TRIB3 with its protein targets, and specificallywith Akt. Posttranslational modifications of TRIB3, yet to bedescribed, and/or changes in the interaction with other membersof the Tribbles family that participate in modulating these inter-actions, are two plausible possibilities that are currently investi-gated in our laboratories.

Our results indicate that TRIB3 plays a central role in themechanism of action of ABTL0812, as it has been proposed forother antitumoral drugs, such as THC in glioma and hepatocel-lular carcinoma tumors (43, 44) or salinomycin in lung cancercells (45). Interestingly, these drugs also induce autophagy, as itdoes ABTL0812, suggesting that TRIB3 upregulation effectivelyprovokes a robust inhibition of the Akt/mTORC1 axis in vivo. THCand salinomycin exert their action by stimulation of endoplasmicreticulum stress and then activating ATF4/CHOP transcriptionfactors that regulate TRIB3 gene. In our study, we identify a newroute that links PPARa/g activation, inhibition of Akt/mTORC1via TRIB3 and cancer cell death.

There are contradictory reports in the literature regarding the roleof TRIB3 in human cancer. Some laboratories reported elevatedTRIB3 mRNA levels which correlated with bad prognosis in colo-rectal cancer (46) and breast cancer patients (47). On the otherhand, high TRIB3 protein levels have been associated with goodprognosis sensitivity to radiotherapy in breast cancer patients (48).As TRIB3 is a very stable protein (49), the discrepancies observedbetween TRIB3 mRNA and protein levels in human breast cancerprognosis might be due to posttranslational modifications, still tobe described, that might modulate the ability of TRIB3 to interactwith other proteins that regulate tumor growth. In this study,however, we show that ABTL0812 treatment induces a robustincrease inTRIB3 transcriptionandmRNAandprotein levels,whichcorrelate with cancer cell death and tumor growth inhibition. Ourresults are in agreement with a recent report showing that geneticinhibition of TRIB3 enhances tumorogenesis in different cancermodels (8), and support a role for TRIB3 as a tumor suppressor.

Hyperactivation of the Akt/mTORC1 axis is observed in themajority of human cancers and blocking this pathway is animportant anticancer strategy (5).Wehave shownhere the activityof ABTL0812 in a panel of cancer cell lines that express high levelsof phosphorylated/active Akt. Importantly, we have preliminaryobserved activity of ABTL0812 in patients. ABTL0812 treatment






impaired Akt phosphorylation in PBMCs from patients includedin the ongoing phase I clinical trial (Fig. 6F). Therefore, Aktphosphorylation will be used as a surrogated pharmacodynamicbiomarker, for estimating the recommended dose for the forth-coming phase II trials. We have preliminarily observed increasedTRIB3 mRNA levels in human PBMCs treated with ABTL0812(Supplementary Fig. S7), suggesting that TRIB3 qPCR analysiscould be also used tomonitor the biologic activity of ABTL0812 inhumans. Finally, and given its uniquemechanismof action, it willbe important to evaluate ABTL0812 in combination with stan-dard chemotherapeutical compounds as well as with other targettherapies. We foresee ABTL0812-based therapies will contributeto improve patients' treatment and quality of life.

Disclosure of Potential Conflicts of InterestJ. Espadaler has ownership interest (including patents) in and is a consultant/

advisory board member for Ability Pharmaceuticals SL. J.M. Lizcano is aconsultant/advisory board member for Ability Pharmaceuticals. No potentialconflicts of interest were disclosed by the other authors.

Authors' ContributionsConception and design: M. Salazar, J. Espadaler, M. G�omez-Ferreria, J. Alf�on,C. Dom�enech, J.M. LizcanoDevelopment of methodology: T. Erazo, M. Lorente, A. L�opez-Plana,P. Mu~noz-Guardiola, P. Fern�andez-Nogueira, G. Fuster, J. Hern�andez-Losa,J.M. LizcanoAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): T. Erazo, M. Lorente, P. Mu~noz-Guardiola,P. Fern�andez-Nogueira, J.A. García-Martínez, P. Bragado, M. Salazar,J. Hern�andez-Losa, J.R. Bayascas, L. Vidal, G. VelascoAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): T. Erazo, M. Lorente, A. L�opez-Plana,P. Fern�andez-Nogueira, J.A. García-Martínez, P. Bragado, M. Salazar,J. Espadaler, M. Cortal, J. Alf�on, C. Dom�enech, J.M. Lizcano

Writing, review, and/or revision of the manuscript: M. Lorente,A. L�opez-Plana, P. Fern�andez-Nogueira, G. Fuster, J. Espadaler, J. Hern�andez-Losa, J.R. Bayascas, M. Cortal, L. Vidal, P. Gasc�on, M. G�omez-Ferreria, J. Alf�on,G. Velasco, C. Dom�enech, J.M. LizcanoAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): J.M. LizcanoStudy supervision: P. Gasc�on, G. Velasco, J.M. Lizcano

AcknowledgmentsThe authors thank Pablo Escrib�a and Xavier Busquets (Universitat Illes

Balears, Spain) for the preliminary identification of ABTL0812 as an anticanceragent (patent number WO/2010/106211) and Giovani Cincilla, EmilianaD'Oria, and Oscar Villaca~nas (Intelligent Pharma SL) for in silico analysis, LuisBotella and Javier Soto (Medalchemy) for the synthesis of ABTL0812, andWashington Biotechnology Inc. for preliminary preclinical studies. The authorsalso thank Victor Yuste for helpful discussions, Cristina Gutierrez for tissueculture assistance, and Servei de Gen�omica i Inform�atica from the UAB.

Grant SupportThis work was supported by grants from the Government of Catalonia

(ACCIO/FINEBT10-1-0047), and from the Spanish Ministry of Economyand Competitiveness (MINECO), (CDTI/PID/IDI-20101630, GenomaEspa~na/INNOCASH/2011196, ENISA/EBT2012/100375, INNPACTO/IPT-2012-0614-010000). J. Alf�on and M. G�omez-Ferreria were funded by Torres-Quevedo grant (MINECO), and M. Cortal by an Inncorpora grant (MINECO).Work in G. Velasco's laboratory was supported by grants from Spanish Ministryof Economy and Competitiveness (MINECO), Fondo Europeo de desarrolloRegional (FEDER) (PI12/02248 and PI15/00339) and Fundaci�on MutuaMadrile~na (AP101042012).

The costs of publication of this articlewere defrayed inpart 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 July 30, 2015; revised November 20, 2015; accepted November 30,2015; published OnlineFirst December 15, 2015.

References1. GuertinDA, Sabatini DM.Defining the role ofmTOR in cancer. Cancer Cell

2007;12:9—22.2. Lizcano JM, Alessi DR. The insulin signalling pathway. Curr Biol 2002;12:

R236–8.3. Hers I, Vincent EE, Tavare JM. Akt signalling in health and disease.

Cell Signal 2011;23:1515–27.4. LoPiccolo J, Blumenthal GM,BernsteinWB,Dennis PA. Targeting the PI3K/

Akt/mTOR pathway: effective combinations and clinical considerations.Drug Resist Updat 2008;11:32–50.

5. Engelman JA. Targeting PI3K signalling in cancer: opportunities, challengesand limitations. Nat Rev Cancer 2009;9:550–62.

6. Rodon J, Dienstmann R, Serra V, Tabernero J. Development of PI3Kinhibitors: lessons learned from early clinical trials. Nat Rev Clin Oncol2013;10:143–53.

7. Hirai H, Sootome H, Nakatsuru Y, Miyama K, Taguchi S, Tsujioka K, et al.MK-2206, an allosteric Akt inhibitor, enhances antitumor efficacy bystandard chemotherapeutic agents or molecular targeted drugs in vitroand in vivo. Mol Cancer Ther 2010;9:1956–67.

8. SalazarM, LorenteM,Garcia-Taboada E, PerezGE,DavilaD, Zuniga-GarciaP, et al. Loss of Tribbles pseudokinase-3 promotes Akt-driven tumorigen-esis via FOXO inactivation. Cell Death Differ 2015;22:131–44.

9. Erazo T, Moreno A, Ruiz-Babot G, Rodriguez-Asiain A, Morrice NA,Espadamala J, et al. Canonical and kinase activity-independent mechan-isms for extracellular signal-regulated kinase 5 (ERK5) nuclear transloca-tion require dissociation of Hsp90 from the ERK5-Cdc37 complex.Mol Cell Biol 2013;33:1671–86.

10. Rodriguez-Asiain A, Ruiz-BabotG, RomeroW, Cubi R, Erazo T, Biondi RM,et al. Brain specific kinase-1 BRSK1/SAD-B associates with lipid rafts:modulation of kinase activity by lipid environment. Biochim BiophysActa 2011;1811:1124–35.

11. Lizcano JM, Alrubaie S, Kieloch A, Deak M, Leevers SJ, Alessi DR. Insulin-induced Drosophila S6 kinase activation requires phosphoinositide3-kinase and protein kinase B. Biochem J 2003;374:297–306.

12. Salazar M, Carracedo A, Salanueva IJ, Hernandez-Tiedra S, Egia A, LorenteM, et al. Cannabinoid action induces autophagy-mediated cell deaththrough stimulation of ER stress in human glioma cells. Autophagy2009;5:1048–9.

13. Klionsky DJ, Abdalla FC, Abeliovich H, Abraham RT, Acevedo-Arozena A,Adeli K, et al. Guidelines for the use and interpretation of assays formonitoring autophagy. Autophagy 2012;8:445–544.

14. Virgin HW, Levine B. Autophagy genes in immunity. Nat Immunol2009;10:461–70.

15. Yang ZJ, Chee CE, Huang S, Sinicrope FA. The role of autophagy in cancer:therapeutic implications. Mol Cancer Ther 2011;10:1533–41.

16. Maggiora GM, Shanmugasundaram V. Molecular similarity measures.Methods Mol Biol 2011;672:39–100.

17. Xiao B, SuM, KimEL,Hong J, ChungHY, KimHS, et al. Synthesis of PPARgactivators inspired by the marine natural product, paecilocin A. Mar Drugs2014;12:926–39.

18. Koo SH, Satoh H, Herzig S, Lee CH, Hedrick S, Kulkarni R, et al. PGC-1promotes insulin resistance in liver through PPAR-alpha-dependent induc-tion of TRB-3. Nat Med 2004;10:530–4.

19. Du K, Herzig S, Kulkarni RN, Montminy M. TRB3: a tribbles homologthat inhibits Akt/PKB activation by insulin in liver. Science 2003;300:1574–7.

20. Galluzzi L, Pietrocola F, Bravo-SanPedro JM, Amaravadi RK, Baehrecke EH,Cecconi F, et al. Autophagy in malignant transformation and cancerprogression. EMBO J 2015;34:856–80.

21. Rovito D, Giordano C, Vizza D, Plastina P, Barone I, Casaburi I, et al.Omega-3 PUFA ethanolamides DHEA and EPEA induce autophagy


Erazo et al.




through PPARg activation in MCF-7 breast cancer cells. J Cell Physiol2013;228:1314–22.

22. Shinohara H, Taniguchi K, Kumazaki M, Yamada N, Ito Y, Otsuki Y, et al. TAnti-cancer fatty-acid derivative induces autophagic cell death throughmodulation of PKM isoform expression profile mediated by bcr-abl inchronic myeloid leukemia. Cancer Lett 2015;360:28–38.

23. Marcilla-Etxenike A, Martin ML, Noguera-Salva MA, Garcia-Verdugo JM,Soriano-Navarro M, Dey I, et al. 2-Hydroxyoleic acid induces ER stress andautophagy in various human glioma cell lines. PLoS One 2012;7:e48235.

24. Holohan C, Van SS, Longley DB, Johnston PG. Cancer drug resistance: anevolving paradigm. Nat Rev Cancer 2013;13:714–26.

25. Sui X, Chen R, Wang Z, Huang Z, Kong N, Zhang M, et al. Autophagy andchemotherapy resistance: a promising therapeutic target for cancer treat-ment. Cell Death Dis 2013;4:e838.

26. Hu M, Huang H, Zhao R, Li P, Li M, Miao H, et al. AZD8055 induces celldeath associatedwith autophagy and activation of AMPK in hepatocellularcarcinoma. Oncol Rep 2014;31:649–56.

27. Zhang H, Berel D, Wang Y, Li P, Bhowmick NA, Figlin RA, et al. Acomparison of Ku0063794, a dual mTORC1 and mTORC2 inhibitor, andtemsirolimus in preclinical renal cell carcinomamodels. PLoSOne 2013;8:e54918.

28. Lin CI, Whang EE, Donner DB, Du J, Lorch J, He F, et al. Autophagyinduction with RAD001 enhances chemosensitivity and radiosensitivitythrough Met inhibition in papillary thyroid cancer. Mol Cancer Res2010;8:1217–26.

29. Li AC, Palinski W. Peroxisome proliferator-activated receptors: how theireffects onmacrophages can lead to the development of a new drug therapyagainst atherosclerosis. Annu Rev Pharmacol Toxicol 2006;46:1–39.

30. Wang L, Waltenberger B, Pferschy-Wenzig EM, Blunder M, Liu X, MalainerC, et al. Natural product agonists of peroxisome proliferator-activatedreceptor gamma (PPARg): a review. Biochem Pharmacol 2014;92:73–89.

31. Ferre P. The biology of peroxisome proliferator-activated receptors: rela-tionship with lipid metabolism and insulin sensitivity. Diabetes 2004;53Suppl 1:S43–S50.

32. Shen B, Chu ES, Zhao G, Man K, Wu CW, Cheng JT, et al. PPARgammainhibits hepatocellular carcinoma metastases in vitro and in mice. Br JCancer 2012;106:1486–94.

33. PanigrahyD, Kaipainen A, Huang S, Butterfield CE, Barnes CM, FannonM,et al. PPARalpha agonist fenofibrate suppresses tumor growth throughdirect and indirect angiogenesis inhibition. Proc Natl Acad Sci U S A2008;105:985–90.

34. Bishop-Bailey D. PPARs and angiogenesis. Biochem Soc Trans 2011;39:1601–5.

35. Li T, Zhang Q, Zhang J, Yang G, Shao Z, Luo J, et al. Fenofibrate inducesapoptosis of triple-negative breast cancer cells via activation of NF-kBpathway. BMC Cancer 2014;14:96.

36. ZangC, LiuH, Bertz J, Possinger K, KoefflerHP, Elstner E, et al. Induction ofendoplasmic reticulum stress response by TZD18, a novel dual ligand forperoxisome proliferator-activated receptor alpha/gamma, in human breastcancer cells. Mol Cancer Ther 2009;8:2296–307.

37. Hegedus Z, Czibula A, Kiss-Toth E. Tribbles: novel regulators of cellfunction; evolutionary aspects. Cell Mol Life Sci 2006;63:1632–41.

38. Grosshans J, Wieschaus E. A genetic link between morphogenesis and celldivision during formation of the ventral furrow in Drosophila.Cell 2000;101:523–31.

39. Hua F, Mu R, Liu J, Xue J, Wang Z, Lin H, et al. TRB3 interacts with SMAD3promoting tumor cell migration and invasion. J Cell Sci 2011;124:3235–46.

40. Ohoka N, Yoshii S, Hattori T, Onozaki K, Hayashi H. TRB3, a novel ERstress-inducible gene, is induced via ATF4-CHOP pathway and is involvedin cell death. EMBO J 2005;24:1243–55.

41. Kiss-Toth E, Bagstaff SM, Sung HY, Jozsa V, Dempsey C, Caunt JC, et al.Human tribbles, a protein family controlling mitogen-activated proteinkinase cascades. J Biol Chem 2004;279:42703–8.

42. Liu J, Zhang W, Chuang GC, Hill HS, Tian L, Fu Y, et al. Role of TRIB3 inregulation of insulin sensitivity and nutrient metabolism during short-term fasting and nutrient excess. Am J Physiol EndocrinolMetab 2012;303:E908–16.

43. Salazar M, Carracedo A, Salanueva IJ, Hernandez-Tiedra S, Lorente M, EgiaA, et al. Cannabinoid action induces autophagy-mediated cell deaththrough stimulation of ER stress in human glioma cells. J Clin Invest2009;119:1359–72.

44. Vara D, Salazar M, Olea-Herrero N, GuzmanM, Velasco G, Diaz-Laviada I.Anti-tumoral action of cannabinoids on hepatocellular carcinoma: role ofAMPK-dependent activation of autophagy. Cell Death Differ 2011;18:1099–111.

45. Li T, Su L, Zhong N, Hao X, Zhong D, Singhal S, et al. Salinomycin inducescell death with autophagy through activation of endoplasmic reticulumstress in human cancer cells. Autophagy 2013;9:1057–68.

46. Miyoshi N, Ishii H, Mimori K, Takatsuno Y, Kim H, Hirose H, et al.Abnormal expression of TRIB3 in colorectal cancer: a novel marker forprognosis. Br J Cancer 2009;101:1664–70.

47. Wennemers M, Bussink J, Scheijen B, Nagtegaal ID, van Laarhoven HW,Raleigh JA, et al. Tribbles homolog 3 denotes a poor prognosis in breastcancer and is involved in hypoxia response. Breast Cancer Res 2011b;13:R82.

48. Wennemers M, Bussink J, Grebenchtchikov N, Sweep FC, Span PN.TRIB3 protein denotes a good prognosis in breast cancer patients and isassociated with hypoxia sensitivity. Radiother Oncol 2011a;101:198–202.

49. Wennemers M, Bussink J, van den BT, Sweep FC, Span PN. Regulation ofTRIB3 mRNA and protein in breast cancer. PLoS One 2012;7:e49439.






2016;22:2508-2519. Published OnlineFirst December 15, 2015.Clin Cancer Res Tatiana Erazo, Mar Lorente, Anna López-Plana, et al. by Upregulating Tribbles-3 PseudokinaseThe New Antitumor Drug ABTL0812 Inhibits the Akt/mTORC1 Axis

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The New Antitumor Drug ABTL0812 Inhibits the Akt/mTORC1 … · Cancer Therapy: Preclinical The New Antitumor Drug ABTL0812 Inhibits the Akt/mTORC1 Axis by Upregulating Tribbles-3