Inhibition of PI3K/Akt/mTOR overcomes cisplatin resistance in the triple negative breast cancer cell line HCC38

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1 Gohr et l. BMC Cncer (2017) 17:711 DOI /s RESEARCH ARTICLE Open Access Inhibition of PI3K/Akt/mTOR overcomes cispltin resistnce in the triple negtive brest cncer cell line HCC38 Kthrin Gohr, Alexndr Hmcher, Lur H. Engelke nd Mtthis U. Kssck * Abstrct Bckground: Widely estblished trgeted therpies directed t triple negtive brest cncer (TNBC) re missing. Clssicl chemotherpy remins the systemic tretment option. Cispltin hs been tested in TNBC but bers the disdvntge of resistnce development. The purpose of this study ws to identify resistnce mechnisms in cispltin-resistnt TNBC cell lines nd select trgeted therpies bsed on these findings. Methods: The TNBC cell lines HCC38 nd MDA-MB231 were subjected to intermittent cispltin tretment resulting in the 3.5-fold cispltin-resistnt subclone HCC38CisR nd the 2.1-fold more resistnt MDA-MB231CisR. Activtion of pro-survivl pthwys ws explored by immunostining of phospho-receptor tyrosine kinses. Trgeted therpies (NVP-AEW541, lptinib nd NVP-BEZ235) ginst ctivted pthwys were investigted regrding cncer cell growth nd cispltin sensitivity. Results: In HCC38CisR nd MDA-MB231CisR, phosphoryltion of epiderml growth fctor receptor (EGFR) nd insulin-like growth fctor 1 receptor (IGF1R) ws observed. In HCC38CisR, tretment with NVP-AEW541 incresed potency of lptinib lmost seven-fold, but both compounds could not restore cispltin sensitivity. However, the dul phosphoinositide 3-kinse (PI3K) nd mmmlin trget of rpmycin (mtor) inhibitor NVP-BEZ235 cted synergisticlly with cispltin in HCC38CisR nd fully restored cispltin sensitivity. Similrly, NVP-BEZ235 incresed cispltin potency in MDA-MB231CisR. Furthermore, NVP-AEW541 in combintion with lptinib restored cispltin sensitivity in MDA-MB231CisR. Conclusion: Simultneous inhibition of EGFR nd IGF1R in cispltin-resistnt TNBC cell lines ws synergistic regrding inhibition of prolifertion nd induction of poptosis. Co-tretment with NVP-BEZ235 or with combintion of NVP-AEW541 nd lptinib restored cispltin sensitivity nd my constitute trgeted tretment option for cispltin-resistnt TNBC. Keywords: Triple negtive brest cncer, HCC38, MDA-MB231, EGFR, IGF1R, NVP-AEW541, NVP-BEZ235, Lptinib, Cispltin resistnce Bckground Brest cncer is the second most common cncer in the world nd the incidence of femle brest cncer hs continuously incresed [1]. In 2013, 1.8 million incident cses of brest cncer occurred, nd the disese cused 464,000 deths [1]. Triple negtive brest cncer (TNBC) ccounts for 10 20% of these brest cncer cses [2]. This type of brest cncer is defined by lcking protein expression of progesterone (PR) nd estrogen receptors (ER) s well s by low ErbB2 expression. For * Correspondence: Mtthis.Kssck@uni-duesseldorf.de Institute for Phrmceuticl nd Medicinl Chemistry, Heinrich Heine University Düsseldorf, Universitätsstrße 1, Düsseldorf, Germny this reson, TNBCs cnnot benefit from endocrine therpies or trstuzumb [3]. Therefore, chemotherpy is the systemic tretment option. The use of cispltin nd crbopltin in tretment of TNBCs is currently investigted in clinicl trils nd initil results indicte beneficil effect for cispltin in neodjuvnt chemotherpy [4, 5]. One mjor chllenge in cispltin therpy is drug resistnce which cn be intrinsic or occur fter severl cycles of therpy. Trigger for cispltin resistnce cn be found pre-trget (e.g. reduced uptke), on-trget (e.g. incresed DNA-repir), post-trget (e.g. inctivtion of TP53) or off-trget [6]. Off-trget mechnisms include ctivtion The Author(s) Open Access This rticle is distributed under the terms of the Cretive Commons Attribution 4.0 Interntionl License ( which permits unrestricted use, distribution, nd reproduction in ny medium, provided you give pproprite credit to the originl uthor(s) nd the source, provide link to the Cretive Commons license, nd indicte if chnges were mde. The Cretive Commons Public Domin Dediction wiver ( pplies to the dt mde vilble in this rticle, unless otherwise stted.

2 Gohr et l. BMC Cncer (2017) 17:711 Pge 2 of 13 of pro-survivl pthwys medited for exmple vi growth fctor receptors. We hve previously shown tht resvertrol or ellgic cid prevented the development of cispltin resistnce in the ovrin cncer cell line A2780. This effect is t lest in prt bsed on the prevention of ctivtion of ErbB2 nd ErbB3 in the course of long-term cispltin tretment [7]. IGF1R ctivtion hs lso been shown to be crucil step in the development of cispltin resistnce [8]. Activtion of growth fctor receptors my lso ply role in the development of cispltin resistnce in TNBC nd due to their involvement in cell prolifertion, poptosis nd metstsis they re considered ttrctive trgets for therpies beyond clssicl chemotherpeutic drugs [9]. In 1998, link between elevted insulin-like growth fctor 1 (IGF1) blood levels nd brest cncer risk in premenopusl women hs been published [10]. In this context the IGF1R emerged s promising trget in cncer therpy. Binding of its lignds to IGF1R results in the ctivtion of minly two downstrem signling networks: PI3K-Akt-mTOR nd RAF-MAPK, both linked to cell survivl nd inhibition of poptosis. Interestingly, not high expression but high phosphoryltion of IGF1R ws predictive for poor prognosis in brest cncer [11]. Extensive reserch in this re ws done but fter initilly promising results, phse III clinicl trils using nti-igf1r-trgeted therpies were minly disppointing [12]. These findings might be due to resistnce mechnisms like compenstory signling vi growth hormone receptors, insulin receptors or epiderml growth fctor receptors. Therefore, combintion therpies were suggested. In vitro studies showed synergistic effect of smll molecule IGF1R inhibitor with gefitinib s EGFR/ ErbB2 inhibitor [13]. However, s hs been seen for IGF1R inhibitors lone, lrger clinicl trils combining IGF1R inhibitors with either gefitinib or erlotinib filed [14]. Tking into ccount tht no biomrkers were used to predict response, predictive tools for the use of IGF1R inhibitors might be necessry. The purpose of our study ws to identify resistnce mechnisms in cispltin-resistnt TNBC cell line leding to trgeted therpies s tretment options in this cncer type. Evlution of the phosphoryltion sttus of receptor tyrosine kinses reveled ctivtion of IGF1R nd EGFR s result of cispltin resistnce. Therefore, inhibitors of these two receptors (NVP-AEW541 nd lptinib) nd n inhibitor of downstrem cting PI3K/ Akt/mTOR (NVP-BEZ235) were evluted regrding their effects on cncer cell growth nd cispltin sensitivity. Indeed, co-tretment of NVP-AEW541 with lptinib incresed potency of lptinib in the cispltinresistnt TNBC cell line HCC38CisR but did not increse cispltin sensitivity. On the other hnd, NVP- BEZ235 cted synergisticlly with cispltin nd fully restored cispltin sensitivity in HCC38CisR. Furthermore, in the highly cispltin-resistnt TNBC cell line MDA-MB231CisR, tretment with NVP-BEZ235 or cotretment of NVP-AEW541 with lptinib incresed potency of cispltin up to 4.8-fold. Methods Mterils NVP-AEW541 nd NVP-BEZ235 were gifts from Novrtis (Bsel, Switzerlnd). Lptinib, KU nd LY were from Cymn Chemicl (Michign, USA). Cispltin ws purchsed from Sigm-Aldrich (Steinheim, Germny). 3-(4,5-Dimethylthizol-2-yl)-2,5- diphenyltetrzolium bromide (MTT) ws purchsed from Serv (Heidelberg, Germny). Propidium iodide ws from PromoCell (Heidelberg, Germny). Roswell Prk Memoril Institute (RPMI) medi 1640, Dulbecco s Modified Egle Medium (DMEM), fetl bovine serum (FBS), penicillin/streptomycin [10,000 U/ml; 10 mg/ml] nd trypsin-edta (0.05% trypsin, 0.02% EDTA in PBS) were purchsed from PAN Biotech (Aidenbch, Germny). Primry ntibodies were purchsed from R&D Systems (Wiesbden, Germny) (pigf1r, IGF1R, p-egfr, EGFR, p-erbb2, ErbB2, p-erbb3, ErbB3) or Snt Cruz Biotechnology (Heidelberg, Germny) (p-akt, Akt, β-actin, PARP). HRP-conjugted secondry ntibodies were from R&D Systems. All other regents nd chemicls were from VWR BDH PROLABO (Drmstdt, Germny). Cell lines nd cell culture The triple negtive brest cncer cell line HCC38 ws obtined from ATCC (Mnsss, USA, ATCC order number: ATCC CRL-2314 ) nd cultivted in RPMI medium supplemented with 10% FBS, 120 μg/ml streptomycin nd 120 U/ml penicillin. The TNBC cell line MDA-MB231 (ATCC, Mnsss, USA, ATCC order number: ATCC HTB-26 ) ws cultivted in DMEM supplemented with 15% FBS, 120 μg/ml streptomycin nd 120 U/ml penicillin. Cells were grown t 37 C in humidified tmosphere contining 5% CO 2. HCC38CisR nd MDA-MB231CisR, the cispltin resistnt subclones of HCC38 nd MDA-MB231, respectively, were generted by intermittent tretment of HCC38 or MDA- MB231 with cispltin for 40 cycles ccording to methods previously published [7, 8, 15]. Cells were grown to 80 90% confluency before using them for ssys. MTT cell vibility ssy Cell vibility ws determined using the MTT ssy s previously described [7]. Resistnce fctor ws clculted s rtio of IC 50 of the resistnt cell line nd IC 50 of the sensitive cell line. To investigte the effect of the smll molecule inhibitors on cispltin cytotoxicity, compounds

3 Gohr et l. BMC Cncer (2017) 17:711 Pge 3 of 13 were dded 48 h prior to 72 h cispltin tretment. For combintion index nlysis, cell vibility ws determined from ech well reltive to the verge bsorbnce of control wells. The combintion indexes (CIs) were clculted using ClcuSyn 2.1 softwre (Biosoft, Cmbridge, U.K.) bsed on the Chou Tlly method [16]. CI > 1 indictes ntgonism. CI = 1 indictes n dditive effect nd CI < 0.9 indictes synergism. Neutrl red cell vibility ssy To exclude compound effects potentilly influencing mitochondril ctivity, neutrl red cell vibility ssy insted of MTT ssy ws performed s previously described [17]. Briefly, fter incubtion time, medium ws removed nd 200 μl neutrl red incubtion solution (medium contining FBS, 0.1 M HEPES buffer ph 7.4 nd 0.01% neutrl red) ws dded. After 2 h, incubtion solution ws removed nd cells were quickly wshed with 1% CCl 2 2H 2 O in 1% formldehyde solution. After second wshing step, cells were lysed with 1:1 mixture of ethnol nd 1% cetic cid. Absorbnce ws mesured t 544 nd 690 nm in FLUOstr microplte reder (BMG Lbtech, Ortenberg, Germny). Doubling time The ssy ws performed s previously described [7]. Cells were seeded in 6-well pltes (Srstedt AG, Nürmbrecht, Germny). After 24, 48, 72, nd 96 h, cells were trypsinized nd wshed with PBS. Totl number of cells in 1 ml buffer ws counted in CyFlow spce (Prtec, Muenster, Germny). Doubling time ws clculted using GrphPd Prism (version 4, GrphPd Softwre Inc., Sn Diego, USA). Western blotting For western blotting, stndrd procedures were used s previously described [7]. RTK signl pthwy nlysis The tyrosine-kinse phospho-proteom ws investigted by humn phospho-receptor tyrosine kinse ntibody rry (Ct# ARY001) from R&D Systems ccording to the mnufcturer s protocol. Cell lyste contining 300 μg protein ws used. Cell cycle nlysis Distribution of cell cycle phses of the different cell lines ws nlyzed by flow cytometry using stndrd procedures s previously described [7]. Apoptosis nlysis Apoptotic cells were determined by propidium iodide stining s previously described [7]. Scrtch ssy Scrtch ssy ws performed ccording to stndrd procedures s previously described [7]. Cell-free re ws determined using ImgeJ [18]. Percentge of spce tht ws occupied with cells fter 24 h ws clculted. Sttisticl nlysis Assys were performed t lest in three independent experiments. Concentrtion effect curves were then generted by nonliner regression curve fitting using the 4-prmeter logistic eqution with vrible hill slope (GrphPd Prism version 4, GrphPd Softwre Inc.). Dt presented re men ± SEM if not otherwise stted. Sttisticl significnce ws ssessed by two-tiled Student s t-test or ANOVA nd considered significnt if p < pic 50 ± SEM leding to the reported IC 50 vlues re shown in Additionl file 1. Results The cispltin resistnt cell line HCC38CisR ws generted by weekly exposure to the IC 50 of cispltin for 6 h. After 40 cycles, the IC 50 (determined by MTT) hs shifted from 2.7 μm to 9.4 μm corresponding to resistnce fctor of 3.5 (Fig. 1). This resistnce fctor is in the rnge of previously reported resistnce fctors of cell lines estblished from cncer ptients before nd fter chemotherpy [19]. Resistnce could be mintined without further cispltin tretment. IC 50 of cispltin vried throughout the durtion of these studies between 7 nd 12 μm for HCC38CisR. HCC38CisR ws chrcterized in comprison to the prentl cell line HCC38. Phospho-receptor tyrosine kinse ntibody rry ws used to determine receptor ctivtion. Other receptors thn those shown in Fig. 1b (EGFR-fmily, IGF1R) were not differentilly phosphorylted. HCC38 showed s expected no ctivtion of ErbB2 but ctivtion of EGFR nd ErbB3. Cispltin resistnce (HCC38CisR) did not generte ErbB2 ctivtion, while EGFR nd IGF1R showed mrkedly enhnced ctivtion in HCC38CisR (Fig. 1b). In contrst, ErbB3 ctivtion ws diminished in HCC38CisR. These results could be confirmed by western blotting (Fig. 1c nd d). In this ssy, expression nd ctivtion of receptor tyrosine kinses (RTKs) ws estimted in HCC38, HCC38CisR (long-term cispltin stress, 40 intermittent 6 h cispltin tretment), nd HCC38 exposed to short-term cispltin stress (6 h IC 50 of cispltin with 24 h or 1 week recovery). IGF1R nd EGFR phosphoryltion ws incresed fter 6 h cispltin stress nd 24 h recovery, nd in HCC38CisR. If HCC38 treted 6 h with cispltin could recover from cispltin stress for one week, receptor phosphoryltion decresed nerly to the initil stte. Evluting the expression of growth fctor receptors, there ws hrdly ny difference between untreted HCC38 nd HCC38CisR. Akt

4 Gohr et l. BMC Cncer (2017) 17:711 Pge 4 of 13 b c e d Fig. 1 (See legend on next pge.)

5 Gohr et l. BMC Cncer (2017) 17:711 Pge 5 of 13 (See figure on previous pge.) Fig. 1 Chrcteriztion of HCC38 nd cispltin-resistnt HCC38CisR. (A) Weekly exposure of HCC38 with the IC 50 of cispltin for 6 h resulted in the cispltin resistnt subclone HCC38CisR with resistnce fctor of t lest 3.5 (p < 0.001). IC 50 cispltin HCC38: 2.7 μm; IC 50 cispltin HCC38CisR: 9.4 μm. Shown re men +/ SEM, n = 3. b Detil of phospho-rtk-rry displys phosphoryltion sttus of EGFR-fmily nd IGF1R in HCC38 nd HCC38CisR. c Immunostining of expression nd ctivtion of signling kinses. Shown is representtive experiment out of 3. HCC38 cells were treted with 2.5 μm cispltin for 6 h followed by recovery of 24 h or 1 week. Untreted HCC38 nd HCC38CisR served s controls. d Densito metric nlysis of the protein bnds of HCC38 nd HCC38CisR were performed using ImgeJ softwre (NIH). Dt re mens ± SD, n = 3. All vlues hve been normlized to HCC38 control. Sttisticl nlysis ws performed using one-wy ANOVA test (* p < 0.05, ** p < 0.01, nd *** p < 0.001). e Cell prolifertion mesured by flow cytometry-bsed cell counting. Doubling times were 23.6 h in HCC38 nd 16.9 h in HCC38CisR nd were significntly different (*** p < 0.001). Shown re men +/ SEM, n =3 expression nd phosphoryltion ws enhnced in HCC38CisR compred to HCC38 either untreted or short-term treted with cispltin. Long-term cispltin tretment resulting in HCC38CisR further incresed prolifertion rte nd decresed doubling time significntly from 24 h to 17 h s displyed in Fig. 1e. Bsed on ctivtion of EGFR nd IGF1R in HCC38CisR (Fig. 1), the dul EGFR/ErbB2 inhibitor lptinib nd the IGF1R inhibitor NVP-AEW541 were chosen for further experiments. The IC 50 of both inhibitors ws lower in HCC38CisR thn in HCC38 (Fig. 2/ b). The effect ws more pronounced for NVP-AEW541 (5.7 μm vs. 2.3 μm) thn for lptinib (9.2 μm vs. 6.0 μm) (Fig. 2/b). Next, we tested the combintion of both inhibitors. In HCC38, co-incubtion of NVP- AEW541 hd no effect on the IC 50 of lptinib, nd vice vers, coincubtion of lptinib hd no effect on the IC 50 of NVP-AEW541 (Fig. 2/b). However, coincubtion of NVP-AEW541 cused significnt increse in potency of lptinib in HCC38CisR (lmost 7-fold from 6.0 to 0.88 μm, Fig. 2). Vice-vers, coincubtion of lptinib resulted in significntly decresed IC 50 for NVP-AEW541 in HCC38CisR (2-fold from 2.3 to 1.1 μm, Fig. 2b). To confirm the observed effects, synergism studies were performed (Tble 1). Anlysis bsed on the Chou-Tlly method [16] suggested synergistic interction between lptinib nd NVP-AEW541 (combintion indexes CI < 0.9) in HCC38CisR. Since MTT ssy cnnot distinguish between inhibition of prolifertion nd induction of poptosis, we exmined induction of poptosis using propidium iodide nucler stining (Fig. 2c). Both inhibitors were dded lone or in combintion for 48 h in concentrtion of 2 μm. In HCC38 the tretment induced nerly no poptotic cells (Fig. 2c). In HCC38CisR, NVP-AEW541 (1.53 ± 1.42%) nd lptinib (2.59 ± 0.83%) showed similrly nerly no induction of poptosis wheres the combintion of both compounds could hevily induce poptosis (28.7 ± 2.62%, Fig. 2c). The effect of this combintion on cell cycle distribution in HCC38CisR ws then determined using propidium iodide stining (Fig. 2d). Agin, NVP-AEW541 nd lptinib lone or in combintion were dded in concentrtion of 2 μm for 48 h prior to ethnol fixtion. NVP-AEW541 nd lptinib lone hd no significnt effects. In contrst, the combintion of both compounds could reduce the frction of cells in the G 2 /M phse from 25.7% to 14.2% while incresing the frction of cells in G 1 phse from 62.8% to 77.7% (p < 0.001; Fig. 2d). Tretment with lptinib, NVP-AEW541 or their combintion hd no effect on cell cycle distribution in HCC38 (see Additionl file 2). Next, the effect on phosphoryltion of Akt, EGFR nd IGF1R fter 6 h tretment of HCC38CisR with n IC 50 of lptinib or NVP-AEW541 lone or in combintion ws determined by western blotting (Fig. 2e, f). Wheres both compounds lone hd only moderte effects on receptor phosphoryltion, their combintion reduced EGFR nd IGF1R phosphoryltion to greter extent. Interestingly, Akt phosphoryltion ws unffected by either tretment. Since EGFR nd IGF1R were ctivted in cispltinresistnt HCC38CisR, we exmined if the combintion of lptinib nd NVP-AEW541 could restore cispltin sensitivity in HCC38CisR (Fig. 2g). HCC38CisR ws pretreted with the inhibitors 48 h prior to cispltin tretment. The inhibitors lone nd in combintion hd no significnt effect on cispltin sensitivity. In HCC38, the sme ws observed: neither lptinib nor NVP-AEW541 lone nor their combintion hd n effect on cispltin sensitivity (see Additionl file 3). It hs been shown tht cncer cells cn esily switch membrne-bound RTK pthwys upon inhibition of prticulr RTK nd still use the sme downstrem signling pthwys [20]. Further, since neither lptinib nor NVP-AEW541 hd n effect on cispltin sensitivity nd both compounds did not lter Akt phosphoryltion incresed in HCC38CisR (Fig. 2e, f), we tested whether NVP-BEZ235, dul inhibitor of PI3K nd mtor, hd n effect on cispltin sensitivity. Evluting the cytotoxicity of NVP-BEZ235 in HCC38 nd HCC38CisR reveled tht the IC 50 ws lower in HCC38 (9.1 nm) thn in HCC38CisR (69.3 nm) (See Additionl file 4). 48 h pretretment with 20 nm NVP-BEZ235 incresed potency of cispltin in HCC38CisR by fctor of 4 into the rnge of the non-resistnt cell line HCC38 (IC 50 HCC38CisR: 7.9 μm; IC 50 HCC38CisR pretreted with 20 nm NVP-BEZ235: 2.0 μm; Fig. 3). In HCC38, 20 nm NVP-BEZ235 hd no effect on cispltin

6 Gohr et l. BMC Cncer (2017) 17:711 Pge 6 of 13 b c d e g f Fig. 2 (See legend on next pge.)

7 Gohr et l. BMC Cncer (2017) 17:711 Pge 7 of 13 (See figure on previous pge.) Fig. 2 Combintion of lptinib nd NVP-AEW541 is hyper-dditive but not reversing cispltin resistnce in HCC38CisR. Coincubtion with 1.5 μm NVP-AEW541 significntly decresed IC 50 of lptinib in HCC38CisR, wheres this tretment hd no effect in HCC38. b Coincubtion with 2 μm lptinib significntly decresed IC 50 of NVP-AEW541 in HCC38CisR but hd no effect in HCC38. c In HCC38CisR (but not in HCC38), the combintion of NVP-AEW541 nd lptinib significntly induced poptosis in hyper-dditive mnner (***p < 0.001). NVP-AEW541 nd lptinib were used t 2 μm. Cells were treted for 48 h nd the mount of poptotic nuclei in the control ws subtrcted from treted smples. d Effect of NVP-AEW541 or lptinib (2 μm, respectively) on cell cycle in HCC38CisR. Combintion of 2 μm NVP-AEW541 nd 2 μm lptinib significntly (***p < 0.001) incresed cell popultion in G 1 (77.7 ± 1.2% vs ± 1.4%) while reducing cell popultion in G 2 /M phse (14.2 ± 1.5% vs ± 1.6%). Incubtion time ws 48 h. e Western blot nlysis of p-egfr, p-igf1r, nd p-akt upon tretment of HCC38CisR with n IC 50 of lptinib or NVP-AEW541 or both compounds for 6 h. f Densitometric nlysis of the protein bnds for p-akt, p-egfr, nd p-igf1r of HCC38CisR were performed using ImgeJ softwre (NIH). Dt re mens ± SD, n = 3. All vlues hve been normlized to untreted HCC38 CisR. Sttisticl nlysis ws performed using one-wy ANOVA test (* p < 0.05). g Effect of 1 μm lptinib nd 1.5 μm NVP-AEW541 on cispltin sensitivity either lone or in combintion. Lptinib nd/or NVP-AEW541 were dded 48 h prior to cispltin tretment. IC 50 of cispltin did not significntly differ. All dt shown re men +/ SEM, n = 3, except (e) showing representtive experiment out of 3 sensitivity (Fig. 3). However, NVP-BEZ235 hd more pronounced effect on cell vibility in HCC38 s observed by reduction of the top plteu of the concentrtion effect curve to 48% in HCC38 versus 74% in HCC38CisR (Fig. 3). To corroborte the observed effect in HCC38CisR, synergism studies were performed. The clculted CIs indicted synergism between cispltin nd NVP-BEZ235 in HCC38CisR (Tble 2). Becuse NVP- BEZ235 inhibits PI3K s well s mtor, we exmined the effect on cispltin sensitivity of compounds inhibiting only one of these trgets: LY ws chosen s PI3K inhibitor, KU s mtor inhibitor (Fig. 3b). 48 h preincubtion with either compound prior to cispltin tretment could significntly (p < 0.001) sensitize HCC38CisR for cispltin tretment by fctor of pproximtely 2. If both inhibitors LY nd KU were combined in 48 h preincubtion prior to cispltin tretment in HCC38CisR, the cispltin IC 50 of the prentl cell line HCC38 ws nerly restored (2.9 μm, Fig. 3b). The effect of NVP-BEZ235 on cispltin sensitivity ws slightly, but significntly (p < 0.05) stronger thn the effect of the combintion of KU nd LY Synergism between NVP-BEZ235 nd cispltin ws observed in MTT (Tble 2) nd further verified by western blotting (Fig. 3c) nd poptosis ssy (Fig. 3d). 48 h preincubtion with 20 nm NVP-BEZ235 followed by 6 h tretment with 3 μm cispltin led to mrkedly enhnced ccumultion of cleved poly ADP-ribose Tble 1 Synergism studies between NVP-AEW541 nd lptinib Lptinib [μm] AEW541 [μm] frction ffected <0.2 polymerse (PARP) in HCC38CisR serving s n indictor of cspse 3 ctivtion. Wheres either compound lone could not induce PARP clevge, the combintion of NVP-BEZ235 nd cispltin mrkedly induced PARP clevge. This effect ws not observed in HCC38 (see Additionl file 5). Similrly NVP-BEZ235 could enhnce the number of cispltin-induced poptotic nuclei significntly (hyper-dditive) without hving n own pronounced poptotic effect. Wheres cispltin lone cused 11.4% poptotic nuclei, ddition of NVP-BEZ235 tripled this effect (35.3%). Agin, this effect could not be observed in HCC38 (see Additionl file 6). Since the effect of NVP-BEZ235 on its different trgets is concentrtion-dependent [21], we tested low (20 nm) nd high (280 nm) concentrtion of NVP- BEZ235 on EGFR, IGF1R nd Akt phosphoryltion (Fig. 3e, f) in HCC38CisR. 280 nm NVP-BEZ235 reduced Akt phosphoryltion wheres 20 nm hd no effect. Further, phosphoryltion of IGF1R nd EGFR ws diminished, prticulrly t 280 nm NVP-BEZ235. Cell cycle ws only ffected by 280 nm (but not 20 nm) NVP- BEZ235 in HCC38CisR (Fig. 3g): cells in G 2 /M phse slightly incresed compred to control (28.3% versus 23.6%) ccompnied by slight decrese of cells in G 1 phse (60.0% versus 67.3%; p < 0.05; Fig. 3g). Eventully, we studied effects of the exmined kinse inhibitors NVP-AEW541, lptinib nd NVP-BEZ235 on the migrtory potentil of HCC38CisR by scrtch ssy (Fig. 4). 24 h fter pplying scrtch to untreted cells, Tble 2 Synergism studies between cispltin nd NVP-BEZ235 BEZ235 [nm] cispltin [μm] frction ffected <0.2

8 Gohr et l. BMC Cncer (2017) 17:711 Pge 8 of 13 b c d e g f Fig. 3 (See legend on next pge.)

9 Gohr et l. BMC Cncer (2017) 17:711 Pge 9 of 13 (See figure on previous pge.) Fig. 3 NVP-BEZ235 tretment fully restores cispltin sensitivity in HCC38CisR. 20 nm NVP-BEZ235 dded 48 h prior to cispltin tretment significntly reduced IC 50 of cispltin in HCC38CisR (p < 0.001) but not in HCC38. b 1 μm KU or 5 μm LY or their combintion significntly reduced IC 50 of cispltin in HCC38CisR (p < 0.001). c Western blot nlysis of PARP nd cleved PARP in HCC38CisR used s n indictor of ctive Cspse 3. For combintion of NVP-BEZ235 nd cispltin, 20 nm NVP-BEZ235 ws incubted 48 h prior to ddition of 3 μm cispltin for 6 h. d Induction of poptosis by NVP-BEZ235 nd cispltin. 20 nm NVP-BEZ235 ws incubted 24 h prior to ddition of 5 μm cispltin for 6 h followed by 24 h of recovery. Combintion of NVP-BEZ235 with cispltin incresed poptotic nuclei (35.3 ± 3.7%) compred to cispltin lone (11.4 ± 2.3%) nd NVP-BEZ235 lone (4.6 ± 2.0%) (***p < 0.001). e Western blot nlysis of p-egfr, p-igf1r, nd p-akt in HCC38CisR upon 48 h tretment with 20 nm or 280 nm NVP-BEZ235. f Densitometric nlysis of the protein bnds of p-egfr, p-igf1r, nd p-akt in HCC38 nd HCC38CisR were performed using ImgeJ softwre (NIH). Dt re mens ± SD, n = 3. All vlues hve been normlized to HCC38 control. Sttisticl nlysis ws performed using one-wy ANOVA test (* p < 0.05, ** p < 0.01, nd *** p < 0.001). g Effect of 20 nm or 280 nm NVP-BEZ235 on cell cycle in HCC38CisR. 280 nm NVP-BEZ235 gve slight but significnt (*p < 0.5) reduction of cells in G 1 phse (67.3 ± 1.6% vs ± 0.9% in control) ccompnied by n increse in cells in G 2 /M phse (23.6 ± 1.4% vs ± 0.5% in control). All dt shown re men +/ SEM, n = 3, except (C/E) showing representtive experiment out of 3 61% of the scrtch ws covered by cells (Fig. 4/b). Tretment with ny of the kinse inhibitors reduced migrtion, however only the combintion of 1.5 μm NVP- AEW541 nd 1 μm lptinib showed significnt inhibition of migrtion (Fig. 4/b). To exclude tht inhibition of migrtion ws only due to reduced prolifertion, three different ssys evluting cell vibility were performed using the sme conditions s pplied in the scrtch ssy: MTT ssy, neutrl red ssy, cell count by flow cytometry. Tretment with NVP-AEW541 or lptinib or their combintion did not ffect prolifertion (Fig. 4c). Only NVP-BEZ235 significntly reduced cell prolifertion compred to untreted control (MTT: 82%, neutrl red: 87%, cell count: 81%). However, NVP- BEZ235 did not significntly inhibit migrtion. Lstly, we extended the study of NVP-AEW541, lptinib, NVP-BEZ235 in HCC38 nd HCC38CisR to the TNBC cell line MDA-MB231. Similrly to the genertion of cispltin-resistnt HCC38CisR, we hve generted 2.1-fold more resistnt sub-line nmed MDA-MB231CisR (Additionl file 7A, Tble 3). Similr to HCC38CisR, MDA-MB231CisR displyed ctivted EGFR nd IGR1R (Additionl file 7B). We then tested combintions of dul nd triple combintions of kinse inhibitors nd cispltin by MTT ssy (Tble 3). In ccordnce with the results obtined in HCC38 nd HCC38CisR (Fig. 3), NVP-BEZ235 hd no effect on cispltin potency in MDA-MB231 but reversed the 2.1-fold cispltin resistnce of MDA- MB231CisR (Tble 3). Furthermore, NVP-BEZ235 incresed poptosis induction in combintion with cispltin compred to either compound lone (Additionl file 7C). Wheres the combintion of NVP-AEW541 plus lptinib only prtilly reversed cispltin resistnce in HCC38CisR (Fig. 2g), this combintion not only reversed the 2.1-fold resistnce of MDA-MB231CisR but shifted cispltin potency by fctor of 4.8 beyond the sensitivity of MDA-MB231 (Tble 3). Notbly, similr to the results in HCC38CisR (Fig. 2c), the combintion of NVP- AEW541 nd lptinib showed highly hyper-dditive effect in the induction of poptosis in both MDA- MB231 nd MDA-MB231CisR (Additionl file 7D). Discussion Among brest cncer, TNBC hs poor prognosis due to the lck of trgeted hormone or HER2- directed therpy nd resistnce development ginst clssicl cytosttics including cispltin currently under clinicl investigtion for TNBC [5]. We hve estblished cellulr model of cispltin resistnce in the TNBC cell line HCC38 to study resistnce mechnisms nd identify trgets for overcoming resistnce. The cispltin resistnt cell line lbeled HCC38CisR exhibited incresed ctivtion observed s phosphoryltion of EGFR nd IGF1R (Fig. 1). Incresed RTK phosphoryltion in HCC38CisR ws ccompnied by fster prolifertion (Fig. 1d) nd higher susceptibility to EGFR nd IGF1R inhibition (Fig. 2, b) compred to the prentl cell line HCC38. By immunostining, we could demonstrte tht n increse in RTK phosphoryltion lso occurred in HCC38 fter short-term (6 h) cispltin exposure. However, in contrst to HCC38CisR showing stble cispltin resistnce with permnent EGFR nd IGF1R ctivtion, the shortterm cispltin-induced receptor phosphoryltion in HCC38 nerly vnished fter 1 week of recovery (Fig. 1c). Crosstlk between RTKs s well s the bility of cncer cells to switch between different growth fctor receptor pthwys is well described [9]. Therefore, lptinib nd NVP-AEW541 were selected to inhibit both ctivted RTKs in HCC38CisR simultneously. According to the Cncer Cell Line Encyclopedi [22], EGFR muttions possibly impiring the effect of lptinib re not described for HCC38. Activtion of EGFR nd IGF1R ws observed in HCC38CisR, but RTK ctivtion is rther ssocited with thn cuse of cispltin resistnce in HCC38CisR s we could not restore cispltin sensitivity by inhibition of these RTKs with lptinib nd NVP-AEW541 (Fig. 2g) even

10 Gohr et l. BMC Cncer (2017) 17:711 Pge 10 of 13 Fig. 4 Combintion of lptinib nd NVP-AEW541 but not NVP-BEZ235 inhibits cell migrtion in HCC38CisR mesured by scrtch ssy. Microscopic imges were obtined before (0 h) nd 24 h fter pplying pipet tip-induced scrtch in nerly confluent cell monolyer. Dt shown re one typicl experiment out of three independent experiments. b Averge migrtion, estimted s spce occupied fter 24 h, showed tht only the combintion of 1.5 μm NVP-AEW541 nd 1 μm lptinib significntly reduced cell migrtion (**p < 0.01). c Cell prolifertion ssys pplied under the conditions of (b) (24 h incubtion). Only 20 nm NVP-BEZ235 significntly reduced cellulr prolifertion (n = 3, *p < 0.05) though both compounds were shown to successfully inhibit EGFR nd IGF1R phosphoryltion (Fig. 2e, f ). Notbly, in the highly cispltin-resistnt cell line MDA-MB231CisR (IC μm), we found 4.8-fold resensitiztion for cispltin upon pretretment with NVP-AEW541 nd lptinib (IC μm, Tble 3). We could demonstrte synergy of lptinib nd NVPAEW541 with respect to inhibition of cell vibility (Tble 1) nd poptosis induction (Fig. 2c, Additionl file 7D). Coincubtion with NVP-AEW541 reduced IC50 of lptinib nerly 7-fold (Fig. 1). This effect my be of clinicl importnce s the resulting IC50 of 0.88 μm is lower thn the reported cmx of lptinib (1.7 4 μm) [23, 24]. In cell cycle nlysis we could show tht the combintion of EGFR nd IGF1R inhibition resulted in n increse in cells in G1 phse. This might be one possible mechnism leding to reduced cell prolifertion. These results re in ccordnce with studies performed on drenocorticl crcinoms pplying EGFR nd IGF1R inhibitors [25].

11 Gohr et l. BMC Cncer (2017) 17:711 Pge 11 of 13 Tble 3 IC 50 vlues (μm) from MTT ssys nd corresponding shift fctors (SF) of cispltin lone nd fter 48 h pretretment with 1.5 μm NVP-AEW541, 2 μm lptinib, 20 nm NVP-BEZ235, or 1.5 μm NVP-AEW541 plus 2 μm lptinib, respectively, in MDA-MB231 nd MDA-MB231CisR cells Compound MDA-MB231 MDA-MB231 CisR IC 50 SF IC 50 SF cispltin cispltin + AEW cispltin + lptinib cispltin + BEZ * cispltin + AEW541 + lptinib * * * p < 0.05 Dt re men of 3 experiments Another effect of the combintion of lptinib nd NVP-AEW541 in HCC38CisR is the reduction of cell migrtion (Fig. 4/b) which ws not due to decresed prolifertion s shown by simultneously performed prolifertion ssys (Fig. 4c). Migrtion of cncer cells serves s mrker for invsion nd the potentil to form metstses. As TNBC hs high risk for metstses [26], drugs reducing migrtion my be vluble in treting TNBC. Although the dvntges of combining RTK inhibitors hve been shown severl yers go [27], the in vitro results hve not yet been trnsferred into clinicl benefits [14]. Tking into ccount tht the pproch of combining NVP-AEW541 nd lptinib showed only synergy in HCC38CisR but not in HCC38, it might be of vlue to select tumors ccording to their RTK ctivtion. Our study demonstrtes tht the phosphoryltion sttus of RTKs predicts response to the combintion of lptinib nd NVP-AEW541 (in HCC38CisR nd MDA-MB231CisR) wheres receptor expression showed only mrginl differences between non-responding HCC38 nd responding HCC38CisR. Therefore, the selection of trgeted therpies by receptor phosphoryltion rther thn receptor expression might be n pproch for further studies. Lptinib nd NVP-AEW541 were ineffective to restore cispltin sensitivity in HCC38CisR (Fig. 2g). However, Akt ws stronger phosphorylted in HCC38CisR thn in untreted or short-term (6 h) cispltin-treted HCC38 (Fig. 1c), ssuming n incresed ctivtion in the course of cispltin resistnce development. Lptinib nd NVP- AEW541 did not influence downstrem Akt phosphoryltion (Fig. 2e) suggesting further mechnisms conserving Akt ctivtion [28]. It hs been shown tht dul inhibition of two kinses in IGF1R signling pthwy is superior to pplying only single gents in the TNBC cell line MDA- MB-231 [29]. Therefore, we chose the dul PI3K/mTOR inhibitor NVP-BEZ235 to ddress incresed Akt ctivtion in HCC38CisR. Synergy of NVP-BEZ235 hs lredy been demonstrted for pclitxel in colon cncer cells [30] nd crbopltin in triple negtive brest cncer cell line [31]. Additionlly, NVP-BEZ235 hs lredy proven its bility to enhnce cispltin sensitivity in cispltin resistnt bldder cncer cell lines [32]. In our study, NVP-BEZ235 could fully restore cispltin sensitivity in the cispltin-resistnt TNBC cell line HCC38CisR nd cted synergisticlly with cispltin (Fig. 3/d, Tble 2). Using KU nd LY , we could demonstrte tht it ws not sufficient to inhibit mtor or PI3K lone, respectively, to obtin the NVP-BEZ235-induced effect on cispltin sensitivity (Fig. 3b). Combining KU nd LY nd thereby mimicking the dul inhibition of NVP-BEZ235 incresed the effect of ech compound lone on cispltin sensitivity (Fig. 3b). Nevertheless, NVP-BEZ235 ws slightly more effective thn the combintion of KU nd LY Other studies hve shown tht mtor inhibition might result in only trnsient decrese or even increse of phospho-akt (p-akt) cused by feedbck ctivtion [31, 33]. Thus, these nd our results llow the conclusion tht the combintion of PI3K nd mtor inhibition is preferred over mtor inhibition lone for cispltin sensitiztion. Lstly, synergy between NVP-BEZ235 nd cispltin ws not observed in HCC38 even though Akt showed some ctivtion, however lower thn in HCC38CisR. This indictes tht NVP-BEZ235 enhnces cispltin sensitivity if next to Akt ctivtion upstrem RTKs such s EGFR nd IGF1R re ctivted. Activted RTKs plus ctivted Akt my thus serve s potentil biomrkers for the use of NVP-BEZ235 in combintion with cispltin in TNBC. These results in HCC38CisR were corroborted by dt obtined with MDA-MB231CisR (Tble 3, Additionl file 7). Conclusions Tken together, ctivtion of EGFR nd IGF1R nd their downstrem signling pthwy kinse Akt is ssocited with resistnce induced by long-term tretment with cispltin in the TNBC cell line HCC38 nd in MDA-MB231. Bsed on these results, two pproches for treting cispltin resistnt cell lines re presented: 1) Simultneous inhibition of EGFR nd IGF1R by lptinib nd NVP-AEW541 is highly synergistic nd results in the induction of poptosis. Furthermore, co-tretment with lptinib nd NVP- AEW541 my increse cispltin sensitivity s seen in MDA-MB231CisR. 2) Co-tretment of cispltinresistnt TNBC cell lines with the PI3K/mTOR inhibitor NVP-BEZ235 nd cispltin is synergistic, fully reversed cquired cispltin resistnce, nd my thus constitute trgeted tretment option for cispltinresistnt TNBC.

12 Gohr et l. BMC Cncer (2017) 17:711 Pge 12 of 13 Additionl files Additionl file 1: pic50 vlues nd stndrd error of the men. pic50 vlues, errors, nd IC50 vlues of ll MTT ssys performed in this study re listed. (DOC 70 kb) Additionl file 2: cell cycle distribution. The cell cycle distribution in HCC38 fter tretment with NVP-AEW541, lptinib or both compounds is displyed s br grph. (DOCX 30 kb) Additionl file 3: MTT ssy of combintion of RTK inhibitors with cispltin. Influence of 48 h preincubtion with 1.5 μm NVP-AEW541, 1 μm lptinib or combintion of both compounds on cispltin sensitivity in HCC38. (DOCX 173 kb) Additionl file 4: MTT ssy of NVP-BEZ235. Effect of NVP-BEZ235 on cell vibility determined by MTT ssy. (DOCX 29 kb) Additionl file 5: Western blot of cleved PARP upon NVP-BEZ235 nd cispltin tretment. Western Blot on cleved PARP fter tretment of HCC38 with 20 nm NVP-BEZ235 or 2 μm cispltin or combintion of both compounds. (DOCX 80 kb) Additionl file 6: induction of poptosis in HCC38 upon NVP-BEZ235 nd cispltin tretment. Induction of poptotic nuclei in HCC38 fter tretment with 2 μm cispltin, 20 nm NVP-BEZ235 or combintion of both compounds. (DOCX 25 kb) Additionl file 7: Chrcteriztion of MDA-MB231 nd cispltin-resistnt MDA-MB231CisR. MDA-MB231 nd cispltin-resistnt MDA-MB231CisR cells were chrcterized by MTT ssy, phospho-rtk sttus, nd induction of poptosis upon kinse inhibitor nd cispltin tretment. (DOCX 141 kb) Abbrevitions Akt: Protein kinse B; CI: Combintion index; c mx : Mximum serum concentrtion; EGFR: Humn epiderml growth fctor receptor; ER: Estrogen receptor; ErbB2: Humn epiderml growth fctor receptor 2; ErbB3: Humn epiderml growth fctor receptor 3; ErbB4: Humn epiderml growth fctor receptor 4; HRP: Horserdish peroxidse; IC 50 : Hlf-mximum inhibitory concentrtion; IGF1: Insulin-like growth fctor 1; IGF1R: Insulin-like growth fctor 1 receptor; mtor: mechnistic trget of rpmycin; MTT: 3-(4,5- Dimethylthizol-2-yl)-2,5-diphenyltetrzolium bromide; p-akt: phospho-akt; PARP: Poly ADP ribose polymerse; p-egfr: phospho-egfr; p-erbb2: phospho-erbb2; p-erbb3: phospho-erbb3; p-erbb4: phospho-erbb4; PI3K: Phosphtidylinositol-3-kinse; p-igf1r: phospho-igf1r; PR: Progesterone receptor; RTK: Receptor tyrosine kinse; TNBC: Triple negtive brest cncer Acknowledgements Not pplicble. Funding This work ws funded by grnts from the Bundesministerium für Wirtschft (BMWi) AiF/ZIM project KF UL9 to MUK. The funding body did not interfere nor hd ny objections regrding the design of the study nd collection, nlysis, nd interprettion of dt nd in writing the mnuscript. Avilbility of dt nd mterils All dt generted or nlysed during this study re included in this published rticle nd its supplementry informtion files. In ddition, rw dtsets used nd/or nlysed during the current study re vilble from the corresponding uthor on resonble request. Authors contributions KG conceived the study, designed the experiments, collected nd nlyzed the dt, interpreted the results nd wrote the mnuscript. AH mde substntil contributions to the conception of the study, performed experiments, nlyzed dt, nd ws substntilly involved in writing nd revising the mnuscript. LHE ws involved in dt cquisition nd dt nlysis. MUK conceived the study, designed the experiments, nlyzed the dt, interpreted the results, wrote the mnuscript nd revision of the mnuscript, nd provided finncil support. All uthors red nd pproved the finl mnuscript nd greed to be ccountble for ll spects of the work. Ethics pprovl nd consent to prticipte Not pplicble. Consent for publiction Not pplicble. Competing interests The uthors declre tht they hve no competing interests. Publisher s Note Springer Nture remins neutrl with regrd to jurisdictionl clims in published mps nd institutionl ffilitions. Received: 20 September 2016 Accepted: 19 October 2017 References 1. Globl Burden of Disese Cncer C, Fitzmurice C, Dicker D, Pin A, Hmvid H, Mordi-Lkeh M, MF MI, Allen C, Hnsen G, Woodbrook R, et l. The globl burden of cncer JAMA Oncol. 2015;1(4): Pp A, Cruso D, Tomo S, Rossi L, Zccrelli E, Tomo F. Triple-negtive brest cncer: investigting potentil moleculr therpeutic trget. Expert Opin Ther Trgets. 2015;19(1): Foulkes WD, Smith IE, Reis JS. 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