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Small Molecule Therapeutics

Combinatorial Treatment with mTOR Inhibitorsand Streptozotocin Leads to Synergistic In Vitroand In Vivo Antitumor Effects in Insulinoma CellsJulien Bollard1,2, C�eline Patte1,2, Patrick Massoma2, Isabelle Goddard2,Nicolas Gadot3, Noura Benslama2, Val�erie Hervieu1,2,4,5, Carole Ferraro-Peyret2,4,5,Martine Cordier-Bussat2, Jean-Yves Scoazec6,7, Colette Roche1,2,Thomas Walter1,2,5,8, and C�ecile Vercherat1,2

Abstract

Streptozotocin-based chemotherapy is the first-line chemo-therapy recommended for advanced pancreatic neuroendocrinetumors (pNETs), whereas targeted therapies, including mTORinhibitors, are available in second-line treatment. Unfortunate-ly, objective response rates to both treatments are limited.Because mTOR pathway activation, commonly observed inpNETs, has been reported as one of the major mechanismsaccounting for chemoresistance, we investigated the potentialbenefit of mTOR inhibition combined with streptozotocintreatment in a subset of pNETs, namely insulinomas. To eval-uate the potential of mTOR inhibition in combination withstreptozotocin, we selected four different inhibitors acting atvarious levels of the pathway (everolimus: inhibition ofmTORC1; MK-2206: inhibition of AKT; BKM120: inhibitionof PI3K, mTORC1, and mTORC2; and BEZ235: inhibition of

mTORC1 and mTORC2). Effects on cell viability and apoptosiswere assessed in insulinoma cell lines INS-1E (rat) and MIN6(mouse) in vitro and were confirmed in vivo by using a mousemodel of hepatic tumor dissemination after intrasplenic xeno-graft. In vitro, all four combinations display synergistic effects.These combinations lead to heterogeneous mTOR pathwayinhibition, in agreement with their respective target, andincreased apoptosis. In vivo, tumor growth in the liver wassignificantly inhibited by combining streptozotocin with ever-olimus (P ¼ 0.0014), BKM120 (P ¼ 0.0092), or BEZ235 (P ¼0.008) as compared to each agent alone. These results suggestthat targeting the mTOR pathway in combination with strep-tozotocin could be of potential benefit for insulinomas andpNET patients and thus support further clinical investigations.Mol Cancer Ther; 17(1); 60–72. �2017 AACR.

IntroductionTherapeutic care of advanced pancreatic neuroendocrine

tumors (pNETs) raises challenging clinical questions. Indeed,

these rare and heterogeneous tumors display variable behaviorin term of evolution and response to treatment. First-line treat-ment of advanced pNETs is based on different prognostic factorsand two main strategies are currently described (1): (i) no che-motherapy with a watch-and-wait approach, or somatostatinanalogues (SSA), for nonfunctional pNETs with low proliferativetumors and stable disease at initial diagnosis; (ii) or a "top-down"strategy with a first-line cytotoxic chemotherapy for more aggres-sive pNETs. Streptozotocin (STZ)-based chemotherapy, eitherwith doxorubicin or 5-fluorouracil (5-FU), remains the standardfirst-line chemotherapy showing a 40% response rate (2, 3). Thenovel targeted therapies sunitinib (4) and everolimus (5) haveimproved the progression-free survival in two phase III clinicaltrials, but the objective response rate did not exceed 10%. Newtherapeutic strategies need thus to be developed in order toimprove the management of patients with unresectable pNETs.

In this context, combination therapies appear as promisingoptions. Recent studies have evaluated the potential of such combi-natorial treatments in neuroendocrine tumors, including pNETs.mTOR inhibitors (everolimus or temsirolimus) were combinedwith SSAs in RADIANT-2 study (6) or with the anti-VEGF antibodybevacizumab (7). However, to date, limited numbers of phase I/IIstudies have evaluated the combination of mTOR inhibitorwith chemotherapy (8). Nevertheless, given (i) the low numberof patients, (ii) the problem of safety when combining two typesof treatment with their respective toxicity, and (iii) the growingnumber of different combinatorial or sequential strategies to be

1Groupe des tumeurs neuroendocrines, D�epartement de Recherche Translation-nelle et Innovation, Centre L�eon B�erard, Lyon, France. 2INSERM U1052/CNRSUMR5286/Universit�e de Lyon, Lyon1 UMR-S1052, Centre de Recherche enCanc�erologie, Lyon, France. 3Plateforme Anatomopathologie-Recherche,D�epartement de Recherche Translationnelle et Innovation, Centre L�eon B�erard,Lyon, France. 4Service Central d'Anatomie et Cytologie Pathologiques, HospicesCivils de Lyon, Hôpital Edouard Herriot, Lyon, France. 5Universit�e de Lyon,Universit�e Claude Bernard Lyon 1, Villeurbanne cedex, France. 6Service depathologie morphologique et mol�eculaire, D�epartement de biologie et patho-logiem�edicales; AMMICa, InsermUS23/CNRSUMS3655, GustaveRoussyCancerCampus, Villejuif, France. 7Facult�e de M�edecine de Bicêtre, Universit�eParis Sud, Universit�e Paris Saclay, Le Kremlin-Bicêtre, France. 8Serviced'h�epatogastroent�erologie et d'oncologie digestive, Hospices Civils de Lyon,Hôpital Edouard Herriot, Lyon, France.

Note: Supplementary data for this article are available at Molecular CancerTherapeutics Online (http://mct.aacrjournals.org/).

T. Walter and C. Vercherat are co-senior authors of this article.

Corresponding Author: C�ecile Vercherat, Centre L�eon B�erard, NeuroendocrineTumor Group, Cheney B 3rd floor, 28 rue Laennec, Lyon 69008, France. Phone:334-6985-6133; Fax: 0033478782955 E-mail: [email protected]

doi: 10.1158/1535-7163.MCT-17-0325

�2017 American Association for Cancer Research.

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evaluated, it is crucial to conduct studies with strong rationale andto rely on in vitro and in vivomechanistic studies, in order to betterunderstand the benefit of combinations, evaluate their tolerance,and choose the best agents.

For future studies, knowledge about mechanisms of resistanceshould be taken in consideration when designing a new thera-peutic approach. On the one hand, chemotherapy has cytotoxiceffects which can be diminished by the activation of specificsurvival and proliferation signaling pathways, leading to resis-tance. On the other hand, targeted therapies can inhibit theseabnormally activated pathways; however, they do not necessarilyhave a cytotoxic effect, leading to stabilization but low objectiveresponse. Based on these observations, we hypothesized thattargeted therapies could prevent the activation of survival path-ways and ultimately suppress chemoresistance.

Even if streptozotocin-based chemotherapy is the oldest cyto-toxic chemotherapy used in pNETs, little is known about thefactors leading to resistance to streptozotocin in pNETs patients.O'Toole and colleagues showed that activation of the mTORpathway was correlated with a low response to streptozotocin.Indeed, in tumor tissues, high expression of AKT was correlatedwith a lack of response to streptozotocin. On the opposite,conserved expression of PTEN (PI3K antagonist) was associatedwith response to streptozotocin (9). These observations are inaccordance with in vivo studies in which streptozotocin is mainlyused to induce diabetes in animal models. In pancreatic b cells,specific deletion of Pten gene, as well as expression of constitu-tively active AKT, protects animals from streptozotocin-induceddiabetes and apoptosis (10, 11). It has been widely described thatmTOR pathway activation has a central role in pNETs. At thegenomic level, modifications of mTOR pathway coding genes(PIK3CA, PTEN, TSC2) are involved in approximately 15% ofpNETs (12); moreover, histochemical analyses show that morethan 85% of pNETs display altered expression levels of TSC2,PTEN or both (13).

Based on these data, it appears pertinent to evaluate thecombination of streptozotocin with mTOR pathway inhibitorsin pNETs.

Given the growing number of mTOR inhibitors, we focusedon those that are the most clinically characterized, and thattarget the pathway at different levels: everolimus (mTORC1),MK-2206 (AKT), BKM120 (PI3K), and BEZ235 (PI3K and dualmTORC1 and mTORC2 inhibitor; refs. 14–16). We evaluatedthe effect of their combination with streptozotocin in preclin-ical in vitro and in vivo models. As an in vivo tool, we tookadvantage of a xenograft mouse model developed in the labwhich leads to hepatic dissemination mimicking hepatic metas-tases observed in pNETs patients. All four combinations syn-ergistically diminished cell viability and increased apoptosisin vitro. Moreover, all combinations but streptozotocin/MK-2206, led to significant antitumor responses in xenograftedmice. Our data suggest that combining mTOR inhibitors withstreptozotocin could be a promising therapeutic option foradvanced pNETs patients.

Materials and MethodsCell lines

QGP-1 cells (Japan Health Sciences Foundation, obtained in2006) were routinely cultured in RPMI Glutamax supplementedwith 10% FBS and 1% penicillin/streptomycin. BON cells (kind

gift from B. Mark Evers, Galveston, TX, obtained in 2004) wereroutinely culture in DMEM Glutamax/F-12K medium (1:1) sup-plemented with 10% FBS and 1% penicillin/streptomycin. Ratradio-induced insulinoma, INS-1E cells (ref. 17; kind gift fromC. Wollheim and P. Maechler, Geneva, Switzerland, obtainedin 2007) were routinely cultured in 5 mmol/L glucose RPMIsupplemented with 10% FBS, 10 mmol/L HEPES, 1 mmol/Lsodium pyruvate, 50 mmol/L b-mercaptoethanol, and 1% peni-cillin/streptomycin. MIN6 cells obtained from RIP-Tag mice(kind gift from S. Dalle, Institut de Genomique Fonctionnelle,Montpellier, France, obtained in 2009) were routinely culturedin 5 mmol/L glucose DMEM supplemented with 15% FBS,50 mmol/L b-mercaptoethanol, and 1% penicillin/streptomycin.

All cell lines were not authenticated and were obtained frominvestigators who generated them. Upon reception, cells wereamplified and early-passaged stocks were constituted. Cells werepassaged for fewer than three months after thawing.

Cells were tested for the presence of mycoplasma (MycoAlertMycoplasma Detection Kit; Lonza) on a regular basis (once amonth for in vitro experiments, and one day before injections inanimals).

ReagentsStreptozotocin was purchased from Enzo Life Sciences.

Everolimus was purchased from Selleck Chemicals. BKM120,BEZ235, and MK-2206 were purchased from Active Biochem.For Western blot analysis, we used primary antibodies raisedagainst p-AKT(T308), p-AKT(S473), AKT, P70S6K, p-P70S6K(T389), PRAS40, p-PRAS40(T246), 4EBP1, cleaved caspase-3,p-HistoneH3(S10) (Cell Signaling Technology), p-4EBP1(T45) (Abcam), and tubulin (Sigma). For IHC, we used pri-mary antibodies directed to chromograninA (ImmunoStar)and insulin (DAKO) p-P70S6K (T421/T424) (Santa Cruz Bio-technology Inc.).

In vitro evaluation of the therapeutic potential of drugcombinations

The effect of combinatorial treatmentwas determined using the"Bliss independence test" (18). Based on cell viability results foruncombined drugs, an expected responsewas calculated using theformula: C¼ (STZRþ IR)� (STZR� IR). C represents the expectedresponse, STZR is the difference in cell viability with streptozo-tocin treatment compared to untreated, and IR is the differencein cell viability with mTOR inhibitors treatment compared tountreated. For each combination, the expected response wascompared to experimentally obtained response to calculate theDelta Bliss. Sum of all Delta Bliss for a combination of two drugscorresponds to the Bliss Sum indicating antagonism (Bliss Sum <0), additivism (Bliss Sum ¼ 0), or synergism (Bliss Sum > 0).

In vivo studiesFour-week-old female athymic nude mice obtained from

Envigo (Gannat, France) were housed and bred in the patho-gen-free animal facility "AniCan." Animals were anesthetized(isoflurane) during all surgical procedures. After surgery, animalswere allowed to recover in a sterile atmosphere and were fedad libitum with a sterile diet.

Xenografting procedure was carried out as previously described(19). Briefly, 2.5million INS-1E cells were injected into the spleen,from where they disseminated into the liver through the portalvein to form intrahepatic tumor nodules. To avoid hypoglycemia

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due to insulin secretion from INS-1E cells, drinking water wassupplemented with 10% sucrose. Seven days after cell injection,animals were randomized into treatment and control groups.Streptozotocin and everolimus were injected intraperitoneally at10 and 1.5 mg/kg/day, respectively. MK-2206, BKM120, andBEZ235 were orally administrated at 120, 20, and 45 mg/kg/day,respectively. Control mice were treated with vehicle only. Animalswere weighted daily before treatment and glycemia was measuredthree times a week. All animals were sacrificed after 3 weeks oftreatment. In each animal, liver and pancreas were excised andprepared for histological analysis. Each group of treatment wascomposed of at least five animals and two independent experi-ments were performed.

Statistical analysisAll data were mean values of at least three individual experi-

ments and were expressed as mean � SEM. Statistical analyseswere done using GraphPad Prism 6.05 software (test Mann–Whitney, two-tailed; two-way ANOVA, Tukey multiple compar-isons test).

The rest of materials and methods can be found in Supple-mentary Data.

ResultsSensitivity to streptozotocin is correlated to SLC2A2(GLUT2) expression

Given the poor availability of pNETs cell lines and knowingthat pNETs consist in a heterogeneous group of tumor, wewanted to choose the most appropriate cell line to study

streptozotocin combination with mTOR inhibitors. We evalu-ated the mRNA expression of the GLUT2 transporter (SLC2A2gene), which is essential for streptozotocin incorporationwithin the cell (20, 21). We analyzed the expression of SLC2A2mRNA in the human pNETs cells lines QGP-1 and BON, therat cell line INS-1E, and the mouse cell line MIN6. SLC2A2mRNA was detected in both murine cell lines, whereas itsexpression was faint in BON cells and absent in QGP-1 cells(Fig. 1A). Primers specificity was confirmed on a control mRNAextracted from human pancreatic neuroendocrine tissue. Tocorrelate SLC2A2 expression and streptozotocin sensitivity, weanalyzed the level of apoptosis in these four cell lines byWestern blot analysis, because streptozotocin is a known apo-ptosis inducer (22). Caspase-3 cleavage was observed in INS-1Ecells (3 hours after treatment) and MIN6 cells (6 hours aftertreatment) but not in QGP-1 and BON cell lines (Fig. 1B).Regarding these results, we decided to select the INS-1E cellline for further experiments because its genetic backgroundclosely recapitulates what has been observed in patients.Indeed, we ran a whole genome sequencing (WGS) analysison this cell line and mutations characteristic of pNET (MEN1,DAXX, ATRX, mTOR, PTEN, YY1, TSC2, PI3KCA) were interro-gated. Results showed (i) a deleterious frameshift mutationresulting in a premature stop codon (high pathogenicity) forATRX gene, (ii) an inframe deletion in DAXX (moderate path-ogenicity), (iii) a missense mutation in DAXX (moderatepathogenicity). No variants for YY1 or low impact variants(synonymous variants, variants within introns) were foundfor MEN1, mTOR, PTEN, YY1, TSC2, nor PI3KCA genes (Sup-plementary Table S1). This mutational landscape is in

Figure 1.

Correlation between GLUT2 mRNAexpression (Slc2A2 gene) andstreptozotocin (STZ)-inducedapoptosis. A, Slc2A2 gene expressionwas measured by RT-qPCR.Expression data were normalized tothe expression of housekeeping gene(b-Actin). Experiments were done induplicate and repeated three times. B,Caspase-3 activation followingstreptozotocin treatment is visualizedby Western blot analysis in INS-1E,MIN6, QGP1, and BON cells. a-Tubulinwas used as loading control. Blots arerepresentative of at least threeindependent experiments.

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accordance with what has been published so far for insulinomaand pNETs (12, 23). The MIN6 cell line was used to confirmin vitro results.

A rapid and transient activation of mTOR pathway followsstreptozotocin treatment in vitro

To investigate whether combining mTOR inhibitors withstreptozotocin could be of any benefit for pNETs treatment,we first analyzed the effect of streptozotocin on the mTORpathway in the two selected cell lines. After treatment of INS-1Eand MIN6 cell lines, the mTOR pathway activation was mon-itored by evaluating the phosphorylation level of its major

effectors AKT, P70S6K, and 4EBP1 at different time points, byWestern blot analysis. In INS-1E cells, we noticed a quickactivation of the mTOR pathway from 10 minutes to 2 hoursafter incubation with streptozotocin. We detected an increasedphosphorylation of AKT, first on the S473 residue and after1 hour on T308 residue (Fig. 2B). This activation of AKT wasalso correlated with the phosphorylation of p70S6K at T389residue mainly after 1 hour, probably through the activationof mTORC1. However, there was no evidence of activated4EBP1, another target of mTORC1 (Fig. 2B). Results for MIN6cells show a similar activation after 2 hour of treatment,although more modest (Fig. 2C).

Figure 2.

Effect of streptozotocin (STZ)treatment on the mTOR pathway.A, Inhibitors used in this study targetthe mTOR pathway at differentlevels. B and C, mTOR pathway andcaspase-3 activation duringstreptozotocin treatment isvisualized by Western-blot. INS-1Ecells (B) and MIN6 cells (C) weretreated with streptozotocin(2 mmol/L).






Figure 3.

Benefit of mTOR pathway inhibition duringstreptozotocin (STZ) treatment in INS-1E cells(A–D) andMIN6 cells (D–H). Cell viability aftertreatment with drugs combination wasassessed with CellTiterGlo assay. Resultsrepresent the percentage of growth inhibitioncompared to untreated cells; they are meanvalues of at least three independentexperiments (five duplicates/experiments).Cells were treated with increasing doses ofstreptozotocin combined with increasingdoses of mTOR pathway inhibitors:everolimus (A–E), MK-2206 (B–F), BKM120(C–G), and BEZ235 (D–H). I, Bliss sum scoresfor combinations in INS-1E and MIN6 cellswere calculated from delta bliss values(Supplementary Figs. S2 and S3).

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This activation was only transient and followed by an inhi-bition of the whole pathway. The mTOR pathway was activatedin untreated cells 4 hours after changing the media whereas, inparallel, it was abrogated in streptozotocin-treated cells (inhi-bition of phosphorylation of AKT, p-P70 and 4EBP1). Inter-estingly, we also noticed that this inhibition of the mTORpathway after prolonged treatment with streptozotocin was

correlated with an increased apoptosis, as indicated by thelevel of cleaved caspase-3.

Combinations of mTOR inhibitors and streptozotocin havesynergistic effects in vitro

Based on data from the literature showing the involvementof the mTOR pathway in the resistance to streptozotocin

Figure 4.

Combinations of streptozotocin (STZ) and mTOR inhibitors affect mTOR pathway activation and increase apoptosis in INS-1E cells. Activation of mTORpathway was assessed 2 hours (A) and 4 hours (B) after treatment with streptozotocin (2 mmol/L), everolimus (10 nmol/L), MK-2206 (1 mmol/L),BKM120 (5 mmol/L), and BEZ235 (100 nmol/L) by Western blot analysis. C, Apoptosis activation was evaluated after 4 hours of treatment by analysisof caspase-3 cleavage by Western blot analysis.






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and on our above data showing the streptozotocin-inducedactivation of the mTOR pathway, we suggest that combiningmTOR inhibitors with streptozotocin could potentiate strep-tozotocin cytotoxicity ability. To evaluate the effect of suchcombinations, we performed a CellTiter-Glo viability test,followed by a Bliss independence test (18). Viability testsshowed that streptozotocin decreased the viability of bothINS-1E and MIN6 cells 24 hours after treatment (Supplemen-tary Fig. S1A). Four different inhibitors were used to target themTOR pathway: everolimus (mTORC1 inhibitor), MK-2206(Pan-AKT inhibitor), BKM120 (Pan-PI3K inhibitor), andBEZ235 (dual mTORC1, mTORC2, and PI3K inhibitor;Fig. 2A). Treatments with these molecules had different effectson INS-1E and MIN6 cell viability (Supplementary Fig. S1B–S1E). We proceeded to all the combinations between strepto-zotocin and these inhibitors at all doses and measured thereduction in cell viability of INS-1E and MIN6 cells (Fig. 3A–Dand Fig. 3E–H, respectively).

From the viability data, we calculated a delta Bliss score (Sup-plementary Figs. S2A–S2D and S3A–S3D). By adding all deltaBliss values, we obtained the Bliss sum to evaluate whether thesecombinations had antagonist (0) effects. For both INS-1E and MIN6 cell lines, all combina-tions were found to be synergistic (Fig. 3A–I). For both cell lines,the strongest synergy was obtained by combining streptozotocinwith BEZ235 (bliss sum 171 for INS-1E and 247 for MIN6)whereas other combinations had different effects on either cellline. For INS-1E, streptozotocin/everolimus and streptozotocin/MK-2206 Bliss sums were superior to that of streptozotocin/BKM120. On the opposite, streptozotocin/everolimus synergywas mild for MIN6 cells, in contrast to the greater synergyobserved for streptozotocin/MK-2206 and streptozotocin/BKM120 combinations.

mTOR inhibitors and combinations have various effects onmTOR pathway in vitro

To better evaluate the effects of these combinations, weanalyzed the mTOR pathway activation during treatments byWestern blot analysis. As expected, the degree of mTOR inhi-bition after 2 hours of treatment was variable according to theinhibitor used, whereas streptozotocin activates mTOR path-way (Fig. 4A for INS-1E and Supplementary Fig. S4A for MIN6).A full inhibition was obtained with BKM120 and BEZ235, bothalone and in combination with streptozotocin. This was con-firmed by a decreased phosphorylation of AKT (T308 andS473) and its target PRAS40 (T246), but also by a loweredphosphorylation of the two mTORC1 effectors: p70S6K (T389)and 4E-BP1 (T45). As expected, everolimus (mTORC1 inhib-itor) inhibited p70S6K and 4E-BP1, but did not affect AKT andPRAS40 phosphorylation. Treatment with everolimus or ever-olimus/streptozotocin even activated AKT on T308 and S473residues. On the opposite, MK-2206, alone or combined withstreptozotocin, exerts an inhibition of AKT phosphorylation

(on both T308 and S473 residues), of p70S6K and of 4E-BP1.At this time point, combining mTOR inhibitors and strepto-zotocin prevented streptozotocin-induced activation of themTOR pathway.

After 4 hours of treatment, the mTOR pathway was fullyinhibited (absence of phosphorylation of AKT, PRAS40,p70S6K, and 4E-BP1) by the combination of streptozotocinand MK-2206, BKM120, or BEZ235. However we still observedphosphorylated AKT (S473) and PRAS40 (T246) with ever-olimus while other inhibitors decreased the phosphorylationlevels of all components of the mTOR pathway (Fig. 4B forINS-1E and Supplementary Fig. S4B for MIN6).

Combinations enhance apoptosis in vitroKnowing that streptozotocin is an apoptotic inducer, we

analyzed the cleavage of caspase-3. Four hours after treatment,everolimus and BEZ235 do not trigger apoptosis. Streptozoto-cin, MK-2206, and BKM120 used as single agents led to acleavage of caspase-3. When combined with streptozotocin,MK-2206, BKM120, and BEZ235 increased the cleavage ofcaspase-3 (Fig. 4C for INS-1E and Supplementary Fig. S4C forMIN6).

Effect of combinations on tumor development in a xenograftmodel

To evaluate the effect of these combinations in vivo, we used amouse model of intrahepatic tumor dissemination after intras-plenic xenograft of INS-1E cells. Mice were treated with strepto-zotocin, mTOR inhibitors or a combination. To assess the treat-ment efficacy, we quantified the intrahepatic tumor surface bystaining for the endocrine marker chromogranin A (Fig. 5A–E).Everolimus, MK-2206, BKM120 but not BEZ235 induced a non-significant decrease in tumor surface. These decreases were theresult of a reduction in the mean size of tumor nodules foreverolimus and MK-2206 (Supplementary Fig. S5) and of theirnumber for BKM120 (Supplementary Fig. S6).

When used in combination with streptozotocin, everolimus(P ¼ 0.0014), BKM120 (P ¼ 0.0092), and BEZ235 (P ¼ 0.008),but not MK-2206 led to a significant decrease of tumor surfacecompared to control mice (Fig. 5B–E). The effects of combina-tions containing everolimus were also significantly higher thanwith everolimus or streptozotocin alone (Fig. 5B). The size oftumor nodules was significantly lower after treatment withstreptozotocin/everolimus (P < 0.0001) and streptozotocin/BKM120 (P ¼ 0.0101; Supplementary Fig. S5A and S5C).Streptozotocin/BKM120 also decreased the number of tumornodules as compared to controls (P ¼ 0.0235; SupplementaryFig. S6C). Streptozotocin/everolimus and streptozotocin/BKM120 decreased the number of tumor nodules as comparedwith streptozotocin used as single-agent (P ¼ 0.0498 and0.0072, respectively; Supplementary Fig. S6A and S6C). Inter-estingly, BEZ235 alone did not exert any antitumor effect,whereas it led to a significant decrease in both the tumor

Figure 5.Hepatic tumor nodule development and/or growth in xenografted mice is variously affected by treatment with streptozotocin (STZ) alone or combined tomTOR inhibitors compared to controls. While controls received the vehicle (A), treated animals received streptozotocin (10 mg/kg/day) alone (B) orcombined to everolimus (1.5 mg/kg/day; B), MK-2206 (120 mg/kg/day; C), BKM120 (20 mg/kg/day; D), and BEZ235 (45 mg/kg/day; E) for 3 weeks Tovisualize tumor nodules, liver sections were stained for Chromogranin A (CgA) by IHC (�100). Scale bar: 100 mm. Panels are representative of results obtainedin at least three different experiments which included at least three animals for each condition.






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surface (P ¼ 0.0492, compared to BEZ235 alone; Fig. 5E) andthe number of tumor nodules (P ¼ 0.0253, as compared toBEZ235 alone; Supplementary Fig. S6D) when combined tostreptozotocin.

Glycemia is differently affected by mTOR inhibitorsThe assessment of adverse effects of the different treatments

on animals was performed by monitoring body weight. Nodifference was detected within the different groups of treatment(Fig. 6A–D). Because INS-1E is an insulin-secreting cell lineand as different clinical trials reported that mTOR inhibitorscould lead to hyperglycemia (24–27), the effect of streptozo-tocin and mTOR inhibitors on insulin secretion by INS-1Ecells was checked. To do so, we measured the glycemia ofanimals every 2 to 3 days during the experiment and noticedsignificant differences. Control animals and everolimus- andstreptozotocin-treated animals showed a significant decreasedglycemia between day 7 and day 21 to 25 post-graft. While atday 25, everolimus alone or combined did not significantlymodify glycemia compared to control animals (Fig. 6E). MK-2206/streptozotocin showed significantly higher glycemia thanstreptozotocin (P ¼ 0.0104) or MK-2206 alone (P ¼ 0.0260)treated animals (Fig. 6F). BKM120 alone and BKM120/strep-tozotocin also led to higher glycemia compared with strepto-zotocin-treated animals (P ¼ 0.0196 and 0.0021, respectively)as well as BKM120/streptozotocin compared with control ani-mals (P ¼ 0.0274; Fig. 6G). BEZ235/streptozotocin-treatedanimals also presented higher glycemia compared to control(P ¼ 0.0063) or streptozotocin-treated animals (P ¼0.0075; Fig. 6H).

Moreover, streptozotocin is commonly used as a diabetesinducer in vivo due to its ability to specifically provoke b-celldeath. In this study, streptozotocin was used at very lowdoses compared to protocols for diabetes induction (10 vs.40–80 mg/kg/day). Nevertheless, we checked the absence ofdeleterious effect on b cells by staining pancreatic sectionsfor insulin and by assessing b-cell mass (SupplementaryFig. S7). No significant difference was observed in b-cell massof streptozotocin-treated mice compared to controls.

DiscussionAdvanced pNETs represent a therapeutic challenge; indeed,

even if we can control tumor progression with cytotoxic ortargeted therapies, objective responses are rare and resistance willeventually occur. There is thus a constant and unmet need for newtherapeutic options, especially for patients that failed first- andsecond-line therapies. Up-to-date, only few studies identified

factors that could serve as potential predictive biomarkers forresponse to therapy in pNETs (28, 29), meaning that besidessearching for new targets and new molecules to improve efficacyofmedical care, we need to understand tumor progression as wellas resistance mechanisms. Even if combinatorial strategies seemvery attractive, strong rationales andpreclinical studies are neededto test relevant combinations.

The mTOR pathway is frequently activated in pNETs (12, 13)and it is widely known as a key player in chemoresistance.Indeed, the mTOR pathway promotes cell growth, prolifera-tion, and survival (30). Furthermore, PTEN expression and AKTactivity are associated with chemoresistance in various cancers(31–34) including gastroenteropancreatic NETs (9). Many AKT-driven mechanisms of chemoresistance are described, includingNF-kB activation (31), inhibition of apoptosis through theMDM2-p53 axis (35), inhibition of caspase-mediated apopto-sis (36). In this study, we show that streptozotocin treatmentof insulinoma cell lines leads to rapid and transient activationof the mTOR pathway. This paradoxical activation of a survivalpathway following cytotoxic treatment has been describedin other cell lines (34, 37, 38). Hence, in some human breastcancer cell lines, doxorubicin leads to a rapid and robustactivation of AKT in a PI3K-dependent manner, contributingto cellular resistance to chemotherapy (38). Similar observa-tions were made in gastric cancer cells in response to etoposideand doxorubicin. Moreover, pretreatment of cells with PI3Kinhibitors, prior to chemotherapy, enhanced etoposide anddoxorubicin apoptotic effects (34). Caporali and colleaguesalso showed that such an activation of AKT is observed aftertemozolomide treatment in lymphoblastoid TK6 cell line aswell as in the human colorectal HCT116/3-6 cell line throughan ATR-dependent mechanism (37). These observations sup-port the idea of combining mTOR inhibitors with chemother-apy. Because streptozotocin is the first-line treatment foradvanced pNETs including insulinoma and given that theresponse to streptozotocin is correlated with mTOR pathwayactivation, we decided to combine streptozotocin with mTORinhibitors.

Lately, we observed a growing interest for the development ofmTOR inhibitors. Although first-generation inhibitors targetmTORC1 complex, the ability of mTORC2 to activate AKTencouraged the development of compounds targeting bothmTORC1 and mTORC2, and more globally of dual-inhibitorsalso targeting PI3K (39). To understand the specificity of suchinhibitors, we designed a study allowing the comparison offirst-generation inhibitor, everolimus, currently used for pNETstreatment, to second-generation inhibitors targeting specificallyAKT (MK-2206), PI3K (Pan-class I PI3K inhibitor: BKM120), or

Figure 6.Combination of streptozotocin (STZ) with mTOR inhibitors has different effects on the hepatic tumor surface of xenografted mice and does not leadto hyperglycemia. Animals were treated with streptozotocin (10 mg/kg/day; 5/7 days), everolimus (1.5 mg/kg/day), MK-2206 (120 mg/kg/day), BKM120(20 mg/kg/day), and BEZ235 (45 mg/kg/day), alone or in combination, during 3 weeks. A–D, Total tumor surface was measured and compared tototal hepatic surface after treatment with A. streptozotocin � everolimus (CTL vs. streptozotocin þ everolimus: P ¼ 0.0014; everolimus vs. streptozotocin þeverolimus: P ¼ 0.0269; streptozotocin vs. streptozotocin þ everolimus: P ¼ 0.0088). B, Streptozotocin � MK-2206. C, Streptozotocin � BKM120 (CTL vs.streptozotocin þ BKM120: P ¼ 0.0092; streptozotocin vs. streptozotocin þ BKM120: P ¼ 0.0292). D, streptozotocin � BEZ235 (streptozotocin vs.streptozotocin þ BEZ235: P ¼ 0.008; BEZ235 vs. streptozotocin þ BEZ235: P ¼ 0.0492). E–H, Glycemia was controlled three times per week for thewhole treatment duration with E. Streptozotocin � everolimus. F, Streptozotocin � MK-2206 (streptozotocin vs. streptozotocin þ MK-2206: P ¼ 0.0104;MK-2206 vs. streptozotocin þ MK-2206: P ¼ 0.0260). G, Streptozotocin � BKM120 (CTL vs. streptozotocin þ BKM120: P ¼ 0.0274; streptozotocin vs.BKM: P ¼ 0.0196 and streptozotocin vs. streptozotocin þ BKM120: P ¼ 0.0021). H, Streptozotocin � BEZ235 (CTL vs. streptozotocin þ BEZ235: P ¼ 0.0063;streptozotocin vs. streptozotocin þ BEZ235: P ¼ 0.0075; n ¼ at least nine for each group of animal; two-way ANOVA).






both mTORC1/2 and PI3K (BEZ235; Fig. 2A). Logically, inhi-bition of mTOR pathway in vitro is variable. As expected,everolimus inhibits mTORC1 but not AKT which is activatedin a robust manner, probably through loss of the negativefeedback loop exerted by mTORC1 on AKT (40, 41). However,although everolimus seems to have mild effects in vitro (mod-erate synergy with streptozotocin, no or mild apoptosis whenused as single-agent or combined with streptozotocin), it is veryefficient in our preclinical xenograft model; probably due toantitumor direct effects as well as on the microenvironmentand angiogenesis regulation. These results are encouraging;however, sustained activation of AKT suggests that synergycould be even stronger by suppressing this AKT pro-survivalsignaling. Indeed, on the one hand, in vitro assays show verystrong effects of AKT inhibitor MK-2206 (global inhibition ofthe mTOR pathway and strong synergy with streptozotocin oncell viability and apoptosis). On the other hand, in vivo resultsare rather disappointing with a nonsignificant decrease oftumor surface when used as single-agent but no additionalbenefit when MK-2206 is combined to streptozotocin. MK-2206 is currently being evaluated in phase II clinical trials andgave disappointing results (only 6 of 33 stable diseases) in aphase I study in patients with solid tumors. However, amongthe three pNETs patients included in this study, two of thempresented a stabilization of the disease (42). Nevertheless,another study focusing on pNETs reported limited responseto MK-2206 alone (43). In preclinical models of ovarian andlung cancers, the combination of the AKT inhibitor with theEGFR inhibitor erlonitib or the dual EGFR/HER2 inhibitorlapatinib showed antitumor efficacy (14). However, our studysuggests that a better in vivo characterization of the moleculeefficacy might be necessary. It could be interesting to furtherstudy differences in the whole pathway inhibition to under-stand the inability of MK-2206 to synergize with streptozotocinin vivo. Knowing that MK-2206 is a specific inhibitor of AKT(Fig. 2A), we can hypothesize that inhibition of mTORC1and/or mTORC2 is necessary to reach synergy with streptozo-tocin in vivo.

To inhibit the whole mTOR pathway, we also evaluated theeffect of BEZ235. In vitro inhibition of the pathway seems veryefficient and global. However, when used as single-agent in vivo,BEZ235 does not exert any antitumor effect. This puzzlingresult is in accordance with two clinical trials including pNETspatients. The first trial was conducted with patients presentingresistance to everolimus and the second with patient na€�ve formTOR inhibition. Unfortunately, both trials were prematurelystopped due to unmet statistical endpoint (44). Our data are inaccordance with these results as BEZ235 monotherapy does notshow any antitumor effect in vivo. However, we observed thatthe combination of BEZ235 with streptozotocin leads to asignificant antitumor effect in this xenograft model. The efficacyof BEZ235 was demonstrated both in vitro and in vivo inhepatocellular carcinoma (45) and in the human NET cell linesBON and QGP-1 (46). Both cell lines were not used in ourstudy as BON cell line is very heterogeneous and subclonalmodifications could lead to the loss of neuroendocrine tumorscharacteristics (47–49). Therefore, the use of this cell line as amodel of pNETs is controversial; moreover, we observed thatBON cells lead to Mixed Adenoneuroendocrine Carcinoma(MANEC) tumors rather than pure pNETs in our model ofxenograft (unpublished personal observations). Regarding

QGP-1 cells, we noticed that they were not sensitive to strep-tozotocin combined or not to mTOR inhibitors. Because wefailed to detect SLC2A2, the glucose transporter responsible forstreptozotocin entrance in the cell, in QGP-1 cells, we hypoth-esized that streptozotocin was not able to penetrate into thesecells, leading to intrinsic resistance to streptozotocin-inducedcell death. Given the promising results of BEZ235 in combi-nation with streptozotocin in in vitro and in vivo preclinicalstudies, it could be interesting to test it in combination withstreptozotocin in phase I/II clinical trials, with reduced doses ofBEZ235 in order to get an acceptable toxicity profile forpatients.

Finally, inhibition of PI3K with BKM120 is very efficientwhen used as single agent or combined with streptozotocin.BKM120, a pan-PI3K inhibitor, was shown to display a strongantitumor effect in numerous models including human glio-blastoma cell lines (16). Several clinical trials reported itstherapeutic potential mainly when combined to other com-pounds (26). In our experiments, BKM120 was the mostefficient inhibitor and led to the optimal antitumor response.In conclusion, combination of streptozotocin with mTORinhibitors is a very attractive option to treat pNETs. However,further clinical evaluation is necessary to confirm these resultsand to evaluate the efficiency and safety of such combinationsin pNETs patients. The SEQTOR trial is currently recruitingpatients to evaluate the benefit of such combinations. Moreprecisely it aims at determining what sequence (everolimusfollowed by streptozotocin/50-FU vs. streptozotocin/50-FU fol-lowed by everolimus) is the most beneficial. Our results strong-ly suggest that shutting down the mTOR pathway duringstreptozotocin treatment leads to more potent antitumoreffects, supporting thus the rational for a combinatorial treat-ment or a pretreatment with everolimus prior to streptozotocin.In this setting potential toxicity needs to be considered whencombining or sequencing therapies. Everolimus and streptozo-tocin have different toxicity profiles. Main toxicities of mTORinhibitors are skin rash, hyperglycemia, and mucositis (26, 50),whereas the latest studies using streptozotocin according tocurrent guidelines, showed that renal toxicity of STZ is muchlower than initially reported (3). Preclinical studies may help todetermine the best agents to be combined and to optimizedoses of each drug in combination. Finally, optimal trialdesigns will be needed to determine the best therapeuticprotocol i.e., combination or sequence.

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

Authors' ContributionsConception and design: J.-Y. Scoazec, C. Roche, T. Walter, C. VercheratDevelopment of methodology: T. Walter, C. VercheratAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): J. Bollard, C. Patte, P. Massoma, I. Goddard,N. Gadot, C. Roche, T. Walter, C. VercheratAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): J. Bollard, P. Massoma, T. Walter, C. VercheratWriting, review, and/or revision of the manuscript: J. Bollard, P. Massoma,C. Ferraro-Peyret,M.Cordier-Bussat, J.-Y. Scoazec, C. Roche, T.Walter, C. VercheratAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): C. Patte, P. Massoma, I. Goddard, N. Benslama,V. Hervieu, J.-Y. Scoazec, C. VercheratStudy supervision: J.-Y. Scoazec, T. Walter, C. Vercherat

Bollard et al.





AcknowledgmentsThe authors thank M. Colomb�e and F. Bourguillaut for technical assis-

tance; A. Chappot de la Chanonie, E. Servoz, and V. Martin (Laboratoire desMod�eles Tumoraux) for support with in vivo experiments; B. Mark Evers forBON cell line; C. Wollheim and P. Maechler for INS-1E cell line; and S. Dallefor MIN6 cell line.

The costs of publication of this article were defrayed in part by the paymentof page charges. This article must therefore be hereby marked advertisementin accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received April 13, 2017; revised September 29, 2017; accepted October 12,2017; published OnlineFirst October 19, 2017.

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Bollard et al.




2018;17:60-72. Published OnlineFirst October 19, 2017.Mol Cancer Ther Julien Bollard, Céline Patte, Patrick Massoma, et al. Insulinoma Cells

Antitumor Effects inIn Vivo and In VitroLeads to Synergistic Combinatorial Treatment with mTOR Inhibitors and Streptozotocin

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