combined mek and vegfr inhibition in orthotopic …...pathway may be problematic when it is used...

Cancer Therapy: Preclinical

Combined MEK and VEGFR Inhibition in Orthotopic HumanLungCancerModels Results in Enhanced Inhibition of TumorAngiogenesis, Growth, and Metastasis

Osamu Takahashi1, Ritsuko Komaki1, Paul D. Smith5, Juliane M. J€urgensmeier5, Anderson Ryan5,B. Nebiyou Bekele2, Ignacio I. Wistuba3, J€org J. Jacoby4, Maria V. Korshunova1, Anna Biernacka1,Baruch Erez4, Keiko Hosho1, Roy S. Herbst4, and Michael S. O'Reilly1

AbstractPurpose: Ras/Raf/mitogen-activated protein–extracellular signal-regulated kinase (ERK) kinase (MEK)/

ERK signaling is critical for tumor cell proliferation and survival. Selumetinib is a potent, selective, andorally

availableMEK1/2 inhibitor. In this study, we evaluated the therapeutic efficacy of selumetinib alone or with

cediranib, an orally available potent inhibitor of all three VEGF receptor (VEGFR) tyrosine kinases, in

murine orthotopic non–small cell lung carcinoma (NSCLC) models.

ExperimentalDesign:NCI-H441orNCI-H460KRAS-mutant humanNSCLC cellswere injected into the

lungs of mice. Mice were randomly assigned to treatment with selumetinib, cediranib, paclitaxel, selume-

tinib plus cediranib, or control. When controls became moribund, all animals were sacrificed and assessed

for lung tumor burden and locoregional metastasis. Lung tumors and adjacent normal tissues were

subjected to immunohistochemical analyses.

Results: Selumetinib inhibited lung tumor growth and, particularly at higher dose, reduced locoregional

metastasis, as did cediranib. Combining selumetinib and cediranib markedly enhanced their antitumor

effects, with near complete suppression ofmetastasis. Immunohistochemistry of tumor tissues revealed that

selumetinib alone or with cediranib reduced ERK phosphorylation, angiogenesis, and tumor cell prolif-

eration and increased apoptosis. The antiangiogenic and apoptotic effects were substantially enhanced

when the agents were combined. Selumetinib also inhibited lung tumor VEGF production and VEGFR

signaling.

Conclusions: In this study, we evaluated therapy directed against MEK combined with antiangiogenic

therapy in distinct orthotopicNSCLCmodels.MEK inhibition resulted in potent antiangiogenic effects with

decreasedVEGF expression and signaling. Combining selumetinibwith cediranib enhanced their antitumor

and antiangiogenic effects. We conclude that combining selumetinib and cediranib represents a promising

strategy for the treatment of NSCLC. Clin Cancer Res; 18(6); 1641–54. �2012 AACR.

IntroductionLung cancer is a global problem, and new therapies and

approaches to the treatment of lung cancer are urgentlyneeded (1, 2). The approach to the treatment of non–smallcell lung carcinoma (NSCLC) is currently in transition and

an emerging strategy for the treatment of lung and othercancers is personalized therapy based on the molecularcharacteristics of a tumor from individual patients. Asresearch onmolecular targeted therapy and biomarkers thatpredict the effectiveness of such therapies proceeds, moreeffective treatment strategies for lung cancer are becomingpossible (3). Antiangiogenic therapy and therapies directedagainst growth factors and their associated signaling path-ways have emerged as compelling targets against lungcancer that are now being used in the clinic.

Antiangiogenic therapy, and in particular therapy target-ing the VEGF signaling pathway, has shown promise in thetreatment of lung cancer and bevacizumab, an antibodydirected against VEGF, can improve overall survival when itis combined with carboplatin and paclitaxel for patientswith advanced lung cancer (4). However, when bevacizu-mab was combined with cisplatin and gemcitabine foradvanced NSCLC, progression-free survival was improved

Authors' Affiliations:Departments of 1Radiation Oncology, 2Biostatistics,3Pathology, and 4Thoracic/Head and Neck Medical Oncology, The Uni-versity of Texas MD Anderson Cancer Center, Houston, Texas; and5Department of Oncology, University of Oxford, Oxford, United Kingdom

Note: A. Ryan was formerly affiliated to AstraZeneca, Alderley Park,Macclesfield, United Kingdom.

Corresponding Author: Michael S. O'Reilly, Department of RadiationOncology, The University of Texas MD Anderson Cancer Center, 1515Holcombe Boulevard, Houston, TX 77030. Phone: 713-563-2300; Fax:713-563-2331; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-11-2324

�2012 American Association for Cancer Research.

ClinicalCancer

Research

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on February 11, 2020. © 2012 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from

Published OnlineFirst January 24, 2012; DOI: 10.1158/1078-0432.CCR-11-2324

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but no overall survival advantage was observed (5, 6) norwas there a benefit for overall survival when it was com-bined with cisplatin or carboplatin and etoposide inpatients with extensive stage small cell lung cancer (7).VEGF signaling remains an important target in anticancertherapy because of its role in angiogenesis (8), which isfundamental to tumor growth and spread (9), but it hasbecome apparent that treatmentwith bevacizumab alone orwith systemic chemotherapy may not be sufficient for thetreatment of some advanced lung cancers. Furthermore,toxicities associated with bevacizumab, when it is used withsystemic chemotherapy, have been observed in clinicaltrials in lung cancer patients (10).

Cediranib (AZD2171) is an orally available and highlypotent VEGF receptor (VEGFR)-1, 2, and 3 tyrosine kinaseinhibitor with additional activity against the platelet-derived growth factor-b receptor and c-kit (11–13). Cedir-anib is currently being investigated in clinical trials bothalone and in combination with chemotherapy for a varietyof malignancies. The addition of cediranib to standardchemotherapy for metastatic colorectal cancer was associ-ated with improved progression-free survival but did notimprove overall survival in a phase III trial (14, 15). In aphase III trial of cediranib or lomustine alone and incombination in patients with recurrent glioblastoma, nostatistically significant difference in median progression-free survival of 92 days for the cediranib arm or 125 days inthe combination arm was observed as compared with 82days for lomustine alone (16, 17). Progression-free survivalat 6months was 16% in the cediranib armwhich was lowerthan the 25.8% survival at 6 months reported in the earlierphase II trial of cediranib for recurrent glioblastoma (18).For lung cancer, a phase II/III trial (BR24) in which cedir-anib at a 30 mg dose was combined with carboplatin andpaclitaxel for advanced NSCLC showed improved response

rates and progression-free survival but was terminated earlydue to concerns of toxicity (19, 20). Although the clinicalexperience for cediranib in lung and other cancers doesshow evidence for activity, the results show that it may beprudent to combine it with other biologically targetedtherapeutics for the treatment of lung cancer.

Targeted therapy directed against the epidermal growthfactor receptor (EGFR)with gefitinib (21), erlotinib (22), orcetuximab (23) are currently being used in the clinic for thetreatment ofNSCLCwith improvements inprogression-freesurvival and overall survival in subsets of patients (24).EGFR tyrosine kinase inhibitors have shown single-agentactivity in lung cancer patients whose tumors harbor EGFRmutations (25) but the efficacy of these agents for cancersthat harbor Krasmutations is unclear (26). Although somepatients will experience a durable benefit from these agents,in most cases the improvement in survival can only bemeasured in weeks or months. Furthermore, these agentsmay only be effective in a small percentage of lung cancerpatients and resistance to therapy can develop (27).

To overcome some of the limitations of EGFR and othergrowth factor receptor inhibitors and tomore broadly targetlung cancer growth, therapeutic strategies to target signalingpathways that are downstream of these receptors have beeninvestigated (28). The mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK) that are situateddownstream of Ras and Raf represent attractive therapeutictargets for lung and other cancers. MEK signaling is crucialin the regulation ofmultiple processes, including tumor cellproliferation and survival, in a variety of cancers, includinglung cancer, and its inhibition offers a particularly attractivetherapeutic target (29, 30). Selumetinib (AZD6244 andARRY-142886), a potent, selective, and orally availableMEK1/2 inhibitor (31, 32), has been studied clinically ina variety of cancers, including lung cancer (33) and iscurrently being evaluated in a phase II clinical trial inKRAS-mutant NSCLC. However, the inhibition of MEKsignaling alone may not be sufficient in patients withadvanced lung cancer, and feedback mechanisms in thispathway may be problematic when it is used alone (34).

To determine the efficacy of selumetinib for lung cancergrowing in the lung and to investigate its use for thetreatment of lung cancer with antiangiogenic therapy, wehave studied both monotherapy and combination withtargeted therapy directed against VEGFR signaling withcediranib in orthotopic models of human lung cancer. Wereport the results of this novel combination therapy in ourmurine orthotopic models of human lung cancer thatclosely recapitulate the clinical behavior of lung cancer inhumans (35) using 2 different humanNSCLC cell lines thatharbor mutations in Kras.

Materials and MethodsCell cultures

The human lung adenocarcinoma cell line NCI-H441and the human large cell lung cancer cell line NCI-H460,bothofwhichhaveKRASmutations (36, 37),wereobtained

Translational RelevanceLung cancer is amajor worldwide health problem and

a leading cause of cancer-related death and morbidity.The outcome for patients with lung cancer has notsignificantly changed in over 2 decades and more effec-tive therapies for lung cancer are urgently needed. Weevaluated combinedmitogen-activated protein/extracel-lular signal-regulated kinase (MEK) blockade and angio-genesis inhibition directed against VEGF receptor(VEGFR) signaling in orthotopic models of humannon–small cell lung carcinoma (NSCLC) that closelyrecapitulate clinical patterns of lung cancer progressionand allow for the study of the influence of lung micro-environment upon response to therapy. MEK inhibitionpotentiated the effects of anti-VEGF therapy and inde-pendently inhibited lung tumor angiogenesis, VEGFproduction, and VEGFR signaling. These data providea strong basis for clinical trials combining selumetiniband cediranib in lung cancer patients.

Takahashi et al.

Clin Cancer Res; 18(6) March 15, 2012 Clinical Cancer Research1642




from the American Type Culture Collection. Both cell lineswere molecularly characterized by The University of TexasMD Anderson Cancer Center’s Cell Line CharacterizationShared Resource and determined to be free of Mycoplasmaand pathogenic murine viruses. Cells were maintained inRPMI-1640with 10% fetal bovine serum, sodiumpyruvate,nonessential amino acids, L-glutamine, 2-fold vitaminsolution, and penicillin–streptomycin (Invitrogen) andincubated in an atmosphere of 5% CO2 and 95% air at37�C.

Orthotopic model of human NSCLCSix- to 8-week old male athymic nude mice (Taconic)

were used for experiments in accordance with current reg-ulations and standards of the U.S. Department of Agricul-ture, the U.S. Department of Health and Human Services,the NIH, and The University of Texas MD Anderson CancerCenter. Mice were anesthetized with sodium pentobarbital(50 mg/kg body weight) and placed in the right lateraldecubitus position. A 5-mm skin incision overlying the leftchest wall was made and the left lung was visualizedthrough the pleura. A total of 1 � 106 NCI-H441 cells or5 � 105 NCI-H460 cells (single-cell suspensions, greaterthan 90% viability) in 50 mg of growth factor-reducedMatrigel (BD Biosciences) in 50 mL of Hank’s balanced saltsolutionwere injected into the left lungs of themice throughthe pleura with a 30-gauge needle. After tumor cell injec-tion, thewoundwas stapled and themicewere placed in theleft lateral decubitus position and observed until fullyrecovered.

Drug preparation and treatment schedulesSelumetinib and cediranib (provided by AstraZeneca)

were formulated in vehicle consisting of either 0.5% w/vhydroxypropyl methyl cellulose/0.1% w/v Tween 80 or 1%w/v polysorbate 80, respectively. Paclitaxel (Bristol-MyersSquibb) was dissolved in saline immediately before use.Fourteen days after the implantation of NCH-H441 cells or10 days after the implantation of NCI-H460 cells, when thelung tumors were established,mice (10–13 per group)wererandomly allocated to receive treatment with selumetinib(12.5 or 25 mg/kg twice daily by oral gavage), cediranib (3mg/kg once daily by oral gavage), paclitaxel (200 mg permouse once a week intraperitoneally), selumetinib (25mg/kg twice a day by oral gavage) plus cediranib, or vehiclecontrol. Selumetinib was given twice daily at 8-hour inter-vals and cediranib was given once daily, 4 hours after thefirst daily dose of selumetinib. Treatment was continueduntil the control mice became moribund (55 days for theNCI-H441 model or 19 days for the NCI-H460 model), atwhich point all mice were killed by CO2 inhalation andassessed for lung weight, primary lung tumor volume (P�dlong � dshort

2� 6), and the presence of mediastinal lymphnode disease or distant metastasis. For the NCI-H460mod-el, in which mice also develop chest wall tumors, theaggregate volume of the chest wall tumors was combinedwith the primary lung tumor volume to create the totaltumor volume.

Histologic preparation and immunohistochemical-immunofluorescent staining

Primary lung tumors and adjacent lung tissues wereremoved from all of the mice in every treatment group andfixed with 10% formalin and embedded in paraffin ordirectly frozen inOCT cryoembedding compound and thensectioned and stained with hematoxylin and eosin orimmunoantibodies. Immunostaining for CD31 (rat anti-mouse; Pharmingen) and dual immunofluorescence stain-ing for CD31 and activated VEGFR2-3 (rabbit anti-human;EMD Biosciences) were carried out with frozen tissues asdescribed previously (38). Sections of formalin-fixed, par-affin-embedded tissue specimens were used to assesscleaved caspase-3 (Cell Signaling Technology), Ki-67 (rab-bit a-human; Thermo Scientific), VEGF (goat anti-rat/human; R&D), VEGFR2 (rabbit a-human; Santa Cruz), andphosphorylated mitogen-activated protein kinase (MAPK)44/42 (Erk1/2) (Thr202/Tyr204) (anti-human; SignalStainkit from Cell Signaling) as described previously (38–40).

Quantification of microvessel density, vascular area,Ki-67, caspase-3, and activated extracellularsignal-regulated kinase

For quantification of microvessel density and vasculararea in lung tumors, up to 4 random fields for each tumorsection at �100 magnification (60% center field) were cap-tured after staining with anti-CD31 antibody. Microvesselswere counted and vascular area was calculated using ImagePro software (Media Cybernetics, Inc.). Microvessel densitywas presented as the number of microvessels per field and asthe percentage of vascular pixel area to field pixel area. Thenumber of Ki-67- and activated extracellular signal-regulatedkinase (ERK)–positive nuclei was counted regardless of theimmunointensity in 4 random fields at�100 magnification(60%centerfield). Thenumberof cleaved caspase-3–positivecells was counted in similar fashion but at �200 magnifica-tion.Ki-67 immunoreactivitywas expressed as thepercentageof Ki-67–positive cells to the total tumor cells per field.

H-scoring of VEGF and VEGFR2 immunoreactivityFor semiquantification of VEGF and VEGFR2 immunore-

activity, H-scores were independently generated by 2 of theauthors (O.T. and A.B.) who were blinded as to treatmentgroup as described previously (41), with slightmodification.H-scores were based on findings from up to 4 randomlyselected fields for each tumor section at�100magnification(60% center field). Staining intensity was graded as unde-tectable (0), weak (1), medium (2), or strong (3), and thepercentage of positive cells per field was calculated. Theintensity score and the percentage of positive cells werethen multiplied to give an H-score (possible range, 0–300).

Immunofluorescence quantificationDual fluorescent staining for endothelial cells (CD31,

red), activated VEGFR2/3 (green), and tumor cell nuclei(blue) were completed as described above. The expressionof activatedVEGFR2/3 in tumor-associated endothelial cellswas identified by colocalized yellow fluorescence. The pixel

MEK1/2 and VEGFR Inhibition in a Mouse Model of Lung Cancer

www.aacrjournals.org Clin Cancer Res; 18(6) March 15, 2012 1643




areas of green, blue, red, and yellow were quantified usingImage Pro Plus (Media Cybernetics, Inc.) in up to 4 randomfields for each tumor section at�200magnification. Quan-tification of total activated VEGFR2/3 expression was pre-sented as an index of green area to blue area. ActivatedVEGFR2/3 expression in endothelial cells was presented asan index of yellow area to red area. All of the quantificationdata were presented as mean � SEM.

Statistical analysisData were analyzed by Prism5 software (GraphPad Soft-

ware, Inc.). To analyze immunohistochemical and dual-fluorescent findings, themean value for each tumorwas firstcalculated from captured fields. The Kruskal–Wallis testfollowed by the Mann–Whitney U test were then used toassess differences among the treatment and control groupswith respect to body weight, tumor volume, left lungweight, and quantitative immunohistochemical and dual-fluorescent findings within treatment groups and betweentreatment and control groups. We used the Kruskal–Wallisas a gate-keeping procedure (i.e., a protected testing proce-dure) such that wewould not do any pair-wise comparisonsunless the Kruskal–Wallis test was significant. This proce-dure controls the experiment-wise error rate againstmakingat least one false pair-wise inference in the case that theexperiment-wise null is true. In addition to this protectedprocedure, because multiple comparisons were anticipatedbut use of the Bonferroni correction for all interestingpairwise comparisons would have resulted in too stringenta P value, we considered a P value of less than 0.007 asstatistically significant to account for the multiple compar-

isons. Using a comparison-wise type 1 error rate of 0.007controls the experiment-wise type 1 error rate at 0.10.Differences in the incidence of lymph node metastasis ordistant metastasis were analyzed by the Fisher exact prob-ability tests, and a P value of less than 0.05 was consideredstatistically significant.

ResultsSelumetinib and cediranib block orthotopic humanlung cancer progression in the lung and thorax

To evaluate the therapeutic efficacy of selumetinib, aloneand in combination with cediranib, we used orthotopicmodels of lung cancer with NCI-H441 adenocarcinoma orNCI-H460 large cell human NSCLC cells in nude mice. Alltreatments were well tolerated, with no significant differ-ences among groups inbodyweight. The incidence of tumorformation was 100% after implantation in the left lung forboth models (Tables 1 and 2).

In the NCI-H441 human lung adenocarcinoma model(Fig. 1A and Table 1), lung tumors grew within the left lungand spread within the lung and then to the mediastinum.Treatmentwith selumetinib at both dose levels (12.5 and25mg/kg twice a day) inhibited the growth of primary lungtumors by 71% and 82%, respectively, compared withcontrols. Selumetinib, particularly at the higher dose, wasalso effective in reducing the incidence of mediastinaladenopathy. Cediranib monotherapy also inhibitedprimary lung tumor growth by 68% and the incidence ofmediastinal adenopathy. The antitumor and antimetastaticeffects of each agent were substantially enhancedwhen they

Table 1. Inhibition of tumor growth and metastasis by selumetinib and cediranib in an orthotopic model oflung cancer: NCI-H441 human lung adenocarcinoma model

Treatment group

PaclitaxelSelumetinib

CediranibSelumetinib(25 mg/kg b.i.d)

Variable Vehicle only (200 mg/wk) (12.5 mg/kg b.i.d) (25 mg/kg b.i.d) (3 mg/kg/d) þ cediranib

Body weight, g(range)

31.2 (28.4–25.1) 33.6 (29.5–37.5) 33.1 (21.8–36.8) 32 (26.3–34.5) 31.6 (30.2–36.2) 31.9 (29.5–38.5)

Tumor incidence 13/13 9/9 10/10 11/11 10/10 11/11Left lungweight, mg

560 (300–710) 390 (50–990) 335 (100–710) 280 (100–490)b 245 (90–610)a 150 (90–280)b

Total lungweight, mg

750 (510–900) 580 (220–1140) 515 (290–910) 480 (290–620)a 450 (250–800) 330 (250–480)b

Left lung tumorvolume, mm3

905 (255–1077) 235 (24–1416) 262 (123–628)a 160 (67–628)b 294 (14–760)a 94 (10–193)b

Mediastinaladenopathy

10/13 5/9 5/10 3/11c 4/10 0/11b

NOTE: Data are presented as medians and ranges except for incidence.aP < 0.007.bP < 0.001.cP < 0.05 vs. vehicle.

Takahashi et al.





were combined with a 90% reduction in median primarylung tumor volume, 73% reduction in median left lungweight, and near complete suppression of mediastinallymph node metastasis. Treatment with paclitaxel reducedprimary lung tumor volume by 74% but with only modesteffects upon mediastinal adenopathy that were not statis-tically significant.Similar results were observed in the NCI-H460 human

large cell lung cancer orthotopic model (Fig. 1B, Table 2).In the NCI-H460 model, lung tumors grew within the leftlung and spread within the lung and then to the medi-astinum and also to chest wall of the left hemithorax.Paclitaxel treatment was only marginally effective in theNCI-H460 model, as compared with the NCI-H441 mod-el. Selumetinib, at the lower dose, reduced primary lungtumor volume by 65% and the total tumor volume by71%, as compared with control, but did not substantiallyreduce the incidence of mediastinal lymph node metas-tasis. At the higher dose, selumetinib reduced primarylung tumor volume by 90%, total tumor volume by 92%and decreased the incidence of mediastinal lymph nodemetastasis. Cediranib monotherapy was also efficaciousand reduced primary tumor volume by 78% and totaltumor volume by 84%, but had only modest effect uponmediastinal lymph node metastasis, whereas distantmetastasis was completely inhibited. The antitumor andantimetastatic effects of each agent were substantiallyenhanced when selumetinib and cediranib were com-bined with a reduction in primary lung tumor volume

by 96% and total lung tumor volume by 97% and the nearcomplete suppression of lymph node metastasis.

Selumetinib and cediranib inhibit tumor cellproliferation and increase tumor cell apoptosis in lungtumors

To characterize the mechanism of tumor growth inhibi-tion observed in both of our lung cancer models by selu-metinib and cediranib, lung tumors were subjected toimmuhistochemical analyses. Lung tumors from each ofthe different treatment groups and for each of the 2 lungcancer models were assessed for evidence of tumor cellapoptosis, as determined by staining for cleaved caspase-3 (Fig. 2A). Treatment with paclitaxel marginally increasedtumor cell apoptosis in both models. Apoptosis was signif-icantly increased by selumetinib in a dose-dependent fash-ion in the NCI-H441 (P < 0.001) and in the NCI-H460model (P < 0.007) with an approximate 6- and 3-foldincrease, respectively, at the higher dose. Cediranib treat-ment was also associated with a significant (P < 0.001)increase in lung tumor cell apoptosis, relative to control.The combination of selumetinib and cediranib resulted in afurther increase in tumor cell apoptosis with an 8-foldincrease in the NCI-H441 model (P < 0.001) and a 5-foldincrease (P < 0.001) in the NCI-H460 model.

Tumor cell proliferation for lung tumors from each of thetreatment groups in each of the lung cancer models wasassessed by evaluating Ki-67 expression using immunohis-tochemistry (Fig. 2B). Paclitaxel had virtually no effect upon

Table 2. Inhibition of tumor growth and metastasis by selumetinib and cediranib in an orthotopic model oflung cancer: NCI-H460 human large cell lung cancer model

Treatment Group

PaclitaxelSelumetinib

CediranibSelumetinib(25 mg/kg b.i.d) þ

Variable Vehicle only (200 mg/wk) (12.5 mg/kg b.i.d) (25 mg/kg b.i.d) (3 mg/kg/d) cediranib

Body weight, g(range)

29.9 (24.3–32.2) 29.5 (23.2–33.7) 31.0 (21.2–33.2) 30.6 (25.1–33.3) 30.0 (26.4–33.2) 29.1 (26.9–33.2)

Tumor incidence 10/10 10/10 10/10 10/10 9/9 10/10Left lungweight, mg

410 (310–510) 360 (280–520) 275 (190–310)a 180 (120–230)a 210 (170–380)b 165 (110–190)a

Left lung tumorvolume, mm3

379 (124–644) 254 (199–523) 152 (58–361)b 35 (11–111)a 82 (39–238)a 15 (2–46)a

Total tumorvolume, mm3

805 (323–1649) 524 (218–1470) 232 (162–497)a 66 (25–201)a 131 (89–318)a 27 (2–66)a

Mediastinal nodalmetastasis

10/10 9/10 9/10 4/10c 6/9 1/10a

Distantmetastasis

7/10 4/10 0/10d 0/10d 0/10d 0/10d

NOTE: Data are presented as medians and ranges except for incidence.aP < 0.001.bP < 0.007.cP < 0.05.dP < 0.005 vs. vehicle.






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Figure 1. Antitumor effects of selumetinib, cediranib, and paclitaxel in orthotopic models of human NSCLC in mice. �, P < 0.007 or ��, P < 0.001 versus vehiclecontrol or between groups as indicated (Kruskal–Wallis followed by Mann–Whitney U tests). A, primary tumors in the left lungs of representativemice from each treatment group after implantation of NCI-H441 human lung adenocarcinoma cells (circled), with mean primary tumor lung volumes andleft lungweights. Bars, SEM. B, primary tumors in the left lungs (circled) and chest walls of representativemice fromeach treatment group after implantation ofNCI-H460 human lung large cell cancer cells, with mean total tumor volumes (primary lung tumor þ chest wall tumors) and left lung weights. Bars,SEM. The dotted horizontal line indicates the normal left lung weight.

Takahashi et al.





lung tumor proliferation in the NCI-H441 model and onlymarginally impacted proliferation in the NCI-H60 model.Selumetinib monotherapy significantly inhibited lungtumor proliferation in a dose-dependent manner in bothlung cancer models with a more pronounced antiprolifera-tive effect in theNCI-H460model. Cediranibmonotherapyalso significantly inhibited lung tumor proliferation in bothmodels with a more pronounced effect upon NCI-H441tumors. However, when cediranib and selumetinib werecombined, there was little evidence for enhancement oftheir independent antiproliferative effects examined bypharmacodynamic markers used in these studies.These data show that the antitumor effects of cediranib

and selumetinib in our lung cancer models are mediatedthrough both increased tumor cell apoptosis and decreasedtumor cell proliferation but that the enhanced antitumoractivity of the combination of these agents is mediatedprimarily through increased tumor cell apoptosis.

Selumetinib inhibits lung tumor ERK activationTo assess the effects of treatment upon MEK signaling in

lung tumors, lung tumor tissues were assessed for ERKactivation using immunohistochemistry (Fig. 3). BothNCI-H441 lung adenocarcinoma and NCI-H460 large celllung tumors constitutively expressed and activated ERK(pERK). A 2-fold increase in pERK was observed in theNCI-H460 tumors, as compared with the NCI-H441 lungtumors. Treatment with cediranib partially offset ERK acti-vation for lung tumors in both models with a more pro-nounced in the NCI-H441model that may be related to theexpression of VEGFR2 in lung tumor cells that we havereported previously (42). Treatment with selumetinibresulted in a dose-dependent inhibition of ERK activationfor lung tumors in both lung cancer models. At the higherdose of selumetinib, both alone and in combination withcediranib, the activation of ERK in lung tumors was almostcompletely suppressed in the NCI-H441 and NCI-H460lung cancer models. At the lower dose, treatment withselumetinib reduced pERK expression in both lung cancermodels but to a lesser degree in the NCI-H460 model thanin the NCI-H441 model. These data show that selumetinibtreatment can block ERK activation in lung tumors growingorthotopically but that its effects, particularly at lower dose,vary in different lung tumor models.

Selumetinib inhibits lung tumor angiogenesis withenhanced antiangiogenic effects when combined withcediranibTo assess the impact of treatment with selumetinib and

cediranib alone and in combination for lung cancersgrowing orthotopically on vasculature and angiogenesis,lung tumors were stained for CD31 and microvesseldensity and vascular area were then determined (Fig.4). Treatment with paclitaxel had only a modest effectupon lung tumor angiogenesis which was somewhatmore pronounced in the NCI-H460 model. Cediranibtherapy significantly inhibited lung tumor angiogenesisin both lung cancer models. Selumetinib monotherapy

significantly inhibited lung tumor angiogenesis in bothlung cancer models with reduced microvessel density andvascular area. The antiangiogenic effects selumetinib andcediranib were markedly increased when they werecombined.

Selumetinib, but not cediranib, suppresses theexpression of VEGF in orthotopic lung tumors

To clarify the nature of the antiangiogenic effects oftreatment (Fig. 4), we next evaluated the expression ofVEGF (Fig. 5A) and its receptor VEGFR2 (Fig. 5B) in lungtumors in both of the orthotopic lung cancer models.Treatment with paclitaxel did not impact the expressionof VEGF or VEGFR2 in either lung cancer model. Selu-metinib monotherapy reduced the expression of VEGF ina dose-dependent fashion for the NCI-H441 lung tumorswith a 42% decrease at the lower dose (P < 0.0001) and a62% decrease at the high dose (P < 0.0001), relative tocontrol. In the NCI-H460 lung cancer model, VEGFexpression was also offset in lung tumors after treatmentwith selumetinib with a reduction of VEGF expression ofbetween 20% and 25% as compared with control (P <0.007). Cediranib treatment did not affect lung tumorVEGF expression in either of the lung cancer models.None of the treatment conditions affected expression ofVEGFR2 within the tumor vasculature regardless of whichlung cancer cell line was used. These data show thattreatment with the MEK inhibitor selumetinib can offsetVEGF expression in 2 distinct orthotopic lung cancermodels and suggest that the antiangiogenic effects oftreatment with selumetinib in these models is in partdue to this decreased expression.

Selumetinib and cediranib inhibit activation ofVEGFR2/3 in NCI-H441 and NCI-H460 primary lungtumors and their tumor-associated endothelium

As outlined above, MEK inhibition by selumetinibresults in the suppression of angiogenesis in orthotopicNCI-H441 lung adenocarcinomas and NCI-H460 largecell lung cancers that is due, at least in part, to a decreasedVEGF expression in lung tumors. To further elucidate theeffects of treatment upon VEGF-dependent lung tumorangiogenesis, we characterized VEGFR signaling in bothtumor cells and in the tumor vasculature in lung tumorspecimens using dual immunofluorescent staining (Fig.6). Paclitaxel treatment had little or no effect upon VEGFRsignaling in lung tumor cell or tumor-associated endo-thelial cells in either lung cancer model. Cediranib treat-ment blocked VEGFR signaling in both lung tumor cellsand tumor-associated endothelial cells in both lung can-cer models. Treatment with selumetinib significantlyinhibited VEGFR activation in lung tumor cells andtumor-associated endothelial cells in a dose-dependentfashion in both the NCI-H441 and NCI-H460 lung cancermodels. The most profound effects upon VEGFR activa-tion in lung tumors and their associated vasculatures wereobserved when selumetinib and cediranib were com-bined in both lung cancer models. These data show that






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Figure 2. Apoptotic and antiproliferative effects of selumetinib, cediranib, and paclitaxel in orthotopic models of human NSCLC in mice. Lung tumors werecollected 2 hours after the last dose of selumetinib, 20 hours after the last dose of cediranib, and 6 days after the last dose of paclitaxel. Quantitative valueswere determined in 4 random fields for each tumor. Data are presented as means � SEM for 9 to 13 samples per group. Scale bar, 100 mm. �, P < 0.007;��, P < 0.001 versus vehicle control or between groups as indicated (Kruskal–Wallis followed by Mann–Whitney U tests). A, immunohistochemical analysisof cleaved caspase 3 in tumors frommice implantedwith NCI-H441 or NCI-H460 cells. Representative cleaved caspase-3 staining (brown staining in positivecells) in lung tumors viewed at �200 magnification. Tumor cell apoptosis (number of cleaved caspase-3 positive cells per field) is shown. B,immunohistochemical analysis of Ki-67 staining in tumors from mice implanted with NCI-H441 or NCI-H460 cells. Representative Ki-67 staining of lungtumors (brown) viewed at �100 magnification, 60% center field. Tumor proliferation (% of Ki-67–positive cells per total tumor cells) is shown.

Takahashi et al.





MEK inhibition by selumetinib results in a decrease inVEGFR activation in lung tumors that is associated withan antiangiogenic effect in lung tumors in 2 distinct lungcancer models.

DiscussionThe outcome for patients with advanced lung cancer has

not changed substantially over the past several years butrecent advances show that novel biologically targeted ther-apies can improve the outcomes for subsets of lung cancerpatients. However, it has also become apparent that indi-vidual agents will need to be combined if the outcomes forlung cancers are to bemore broadly improved. In this study,weusedorthotopicmodels of human lung adenocarcinomaand large cell lung cancer that closely mimic clinical pat-terns of lung cancer spread and progression to investigateantiangiogenic therapy directed against VEGFR signalingwith cediranib and molecularly targeted therapy directed

against MEK signaling with selumetinib alone and in com-bination. To our knowledge, this is the first report of theeffects of MEK inhibition with antiangiogenic therapy inmurine orthotopic models of NSCLC. We found that eachagent was effective for the treatment of lung cancer in thesemodels with inhibition of lung tumor growth and, to alesser degree, lymph node metastasis with efficacy superiorto that observed for chemotherapy with paclitaxel. Whenselumetinib and cediranib were combined, a substantialenhancement of their individual antitumor effects wasobserved with improved efficacy within the lung and a nearcomplete suppression of lung cancer progression andmetastasis in both models. Our finding that the combina-tion of these agents impacted both primary tumor andmetastatic growth most effectively has direct clinical rele-vance. Surprisingly, MEK inhibition by selumetinib alsosuppressed lung tumor angiogenesis and targeted bothVEGF production and VEGFR activation in lung tumors,resulting in substantial antiangiogenic effects.

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Figure 3. Effects of selumetinib, cediranib, and paclitaxel upon ERK signaling in orthotopic models of humanNSCLC inmice. Representative pERK staining oflung tumors (purple) and quantified pERK expression (number of pERK positive cells per field). All stains are viewed at �100 magnification, 60%center field. Lung tumors were collected 2 hours after the last dose of selumetinib, 20 hours after the last dose of cediranib, and 6 days after the last dose ofpaclitaxel. Quantitative values were determined in 4 random fields for each tumor. Data are presented as means � SEM for 9 to 13 samples per group.Scale bar, 100 mm. �, P < 0.007; ��, P < 0.001 versus vehicle control or between groups as indicated (Kruskal–Wallis followed by Mann–Whitney U tests).






MEK is an attractive therapeutic target for lung cancertreatment because it is situated downstream of Ras and Raf,which are highly activated in Kras-mutated lung cancer(43). Many Kras-mutant cancer cells have been shown tobe sensitive toMEK inhibitors, (44) and Krasmutations canbe detected in up to 30% of lung cancers, dependent uponhistology and ethnicity (45, 46), suggesting that a subset oflung cancerswould likely be highly sensitive to selumetinib.Our finding that selumetinib was effective in 2 distinct Krasmutant human lung cancer models supports and validatesthis hypothesis. Although monotherapy with selumetinibresulted in antitumor and some antimetastatic effects inboth of our lung cancer models, the antimetastatic effectsweremore apparent in theNCI-H441 lung adenocarcinomamodel. The increased antimetastatic efficacy observed inthis model is associated with differences in the constitutiveexpression and activation of ERK in NCI-H460 and NCI-H441 lung tumors. Both cell lines have KRAS mutationswith activation of ERK for lung tumors from both cell lines.

However, activated ERK was nearly twice as high in NCI-H460 lung tumors, as compared with NCI-H441 lungtumors, and NCI-460 cells are PI3KCa and LKB1 mutant,both of which might provide a degree of resistance to MEKinhibition. In our studies, a lower dose of selumetinibinhibited ERK activation almost completely in the NCI-H441model but by only 46% in the NCI-H460 cells. Thesefindings underscore the importance of MEK signaling inlung cancer progression. However, additional studies areneeded to determine whether molecular profiling of lungcancer specimens could be of use to select patients whomight best benefit from therapy with selumetinib and tohelp tailor the dosing of this agent.

Selumetinib was a potent inhibitor of lung tumor angio-genesis in our orthotopic models, and the addition ofselumetinib to cediranib resulted in amarked enhancementof their individual antiangiogenic effects. Interestingly,selumetinib reduced the production of VEGF in the lungtumors, particularly in the NCI-H441 model. The finding

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Figure 4. Antiangiogenic effects of selumetinib, cediranib, and paclitaxel in orthotopicmodels of humanNSCLC inmice as assessed by immunohistochemicalanalysis of CD31. Representative CD31 staining of lung tumors (brown) viewed at �100 magnification, 60% center field, and microvessel density(number of CD31-positive objects per field) and vascular area (area of CD31-positive objects per field area� 100%) are shown. Lung tumors were collected 2hours after the last dose of selumetinib, 20 hours after the last dose of cediranib, and 6 days after the last dose of paclitaxel. Quantitative valueswere determined in 4 random fields for each tumor. Data are presented as means � SEM for 9 to 13 samples per group. Scale bar, 100 mm. �, P < 0.007;��, P < 0.001 versus vehicle control or between groups as indicated (Kruskal–Wallis followed by Mann–Whitney U tests).

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Figure 5. Effects of selumetinib, cediranib, andpaclitaxel uponVEGFandVEFGRexpression in orthotopicmodels of humanNSCLC inmice. Lung tumorswerecollected 2 hours after the last dose of selumetinib, 20 hours after the last dose of cediranib, and 6 days after the last dose of paclitaxel. Quantitative valueswere determined in 4 random fields for each tumor. Data are presented as means � SEM for 9 to 13 samples per group. Scale bar, 100 mm. �, P < 0.007;��, P < 0.001 versus vehicle control or between groups as indicated (Kruskal–Wallis followed by Mann–Whitney U tests). A, immunohistochemicalanalysis of VEGF in lung tumors frommice implanted with either NCI-H441 or NCI-H460 cells. Representative VEGF staining (brown cytoplasmic staining) inlung tumors and H-scores for VEGF expression are shown. B, immunohistochemical analysis of VEGR2 in lung tumors from mice implanted witheither NCI-H441 or NCI-H460 cells. Representative VEGFR2 staining (brown cytoplasmic staining) in lung tumors and H-scores for VEGFR2 expression areshown.






that MEK inhibits VEGF expression is consistent with stud-ies showing that VEGF expression is downregulated afterEGFR inhibition (47) and provides additional mechanismfor this process. In vitro studies using head and neck cancercell lines show that the VEGF expression after EGFR acti-vation is dependent upon both PI3K and MAPK signaling(48, 49). The MEK inhibitor PD0325901 decreased theexpression of the proangiogenic factors VEGF and interleu-kin 8 in vitro in humanmelanoma cells (50). Prior studies ina murine model of hepatocellular carcinoma showed thatthe antitumor and antiangiogenic effects of rapamycin (51)or sorafenib (52) could be enhanced by the addition ofselumetinib, and that the combination of these agents wasassociated with modest inhibition in VEGFR signaling inliver tumor lysates with reduced circulating levels of VEGF(51). In pancreatic cancer subcutaneous xenograft murinemodels,MEK inhibitionby selumetinib, but not rapamycin,resulted in decreased microvessel density in the subcutane-ous tumors anddecreasedVEGF levels in tumor lysates (53).

Tumor lysates from Calu-6 lung cancer intradermal xeno-grafts in mice-treated selumetinib also showed decreases inVEGF levels (54). From these reports and the findings of thisstudy, we surmise that selumetinib exerts an antiangiogeniceffect in lung tumors by directly and indirectly targetingVEGF and its receptors.

Thedownregulationof VEGF expression thatweobservedin NCI-H441 lung adenocarcinomas after therapy withselumetinib was associated with an inhibition of VEGFRsignaling both in lung tumor cells and the associated lungtumor vasculature and a resultant antiangiogenic effect. Wealso observed a potent inhibition of lung tumor angiogen-esis and inhibition of VEGFR signaling in lung tumor cellsand the associated tumor vasculature in theNCI-H460 largecell lung cancer model. However, in the NCI-H60 model,the expression of VEGF after treatment with selumetinibwas not as dramatic inNCI-H441model. These data suggestthat the downregulation of VEGF alone after treatmentwith selumetinib cannot fully explain the observed





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Figure 6. Effects of selumetinib, cediranib, and paclitaxel upon VEFGR signaling in orthotopic models of human NSCLC in mice. CD31/pVEGFR2/3 dualfluorescent staining in tumors from mice implanted with either NCI-H441 or NCI-H460 cells was completed. Representative colocalized CD31/pVEGFR2/3staining of lung tumors viewed at �200 magnification are shown. Fluorescent red indicates CD31-positive endothelial cells; fluorescent green, totalpVEGFR2/3-positive cells; fluorescent yellow, pVEGFR2/3-positive endothelial cells. Quantification of total activated VEGFR2/3 expression is presented asan index of green area to blue area. Activated VEGFR2/3 expression in endothelial cells is presented as an index of yellow area to red area. All quantificationdata are presented as means � SEM for 9 to 13 samples per group. Scale bar, 50 mm. �, P < 0.007 or ��, P < 0.001 versus vehicle control or betweengroups as indicated (Kruskal–Wallis followed by Mann–Whitney U tests).

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antiangiogenic effects, and that the inhibition of MEK byselumetinib may have both direct and indirect effects uponVEGFR signaling with a resultant multicentric antiangio-genic effect. Prior studies in subcutaneous tumor xenograftand in vitro organotypic angiogenesis assays have shownthat the expression of dominant-negative MEK1 in thetumor vasculature results was associated with antivasculareffects, and that ERK–MAPK signaling promotes endothe-lial cell survival sprouting with downregulation of Rho-kinase activity (55). Further investigation is needed toclarify themechanismbywhich selumetinib inhibits angio-genesis, but our data show that MEK inhibition targetstumor angiogenesis with a multicentric effect.In summary, our study is, to our knowledge, the first

evaluation of therapy directed against MEK in combinationwith anti-VEGF therapy in orthotopic models of NSCLC.MEK inhibition resulted in potent antiangiogenic effects forlung cancers mediated by downregulation of VEGF expres-sion and impaired VEGFR signaling.Wehave further shownthat selumetinib or cediranib can significantly inhibittumor angiogenesis and lung cancer growth and progres-sion with increased tumor cell apoptosis in our orthotopicmodels. Combining selumetinib with cediranib enhanced

their antitumor and antiangiogenic effects, with near-com-plete suppression of lung tumor growth andmetastasis. Weconclude from these findings that the combination ofselumetinib and cediranib represents a promising strategyfor the treatment of NSCLC and provides a strong basis forthe design of clinical trials for this purpose.

Disclosure of Potential Conflicts of InterestP.D. Smith, J.M. J€urgensmeier, and A. Ryan are or were AstraZenenca

employees. R.S. Herbst and M.S. O’Reilly received research funding fromAstraZeneca and formerly served on AstraZeneca Advisory Boards. The otherauthors disclosed no potential conflicts of interest.

AcknowledgmentsThe authors thank Christine F. Wogan of MD Anderson’s Division of

Radiation Oncology for editorial review and comments.

Grant SupportThis work was supported by the NIH through MD Anderson’s Cancer

Center Support grant CA016672 and by a research grant from AstraZeneca.The costs of publication of this article were defrayed in part by the

payment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received September 8, 2011; revised January 1, 2012; accepted January 5,2012; published OnlineFirst January 24, 2012.

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Takahashi et al.




2012;18:1641-1654. Published OnlineFirst January 24, 2012.Clin Cancer Res Osamu Takahashi, Ritsuko Komaki, Paul D. Smith, et al. Angiogenesis, Growth, and MetastasisCancer Models Results in Enhanced Inhibition of Tumor Combined MEK and VEGFR Inhibition in Orthotopic Human Lung

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combined mek and vegfr inhibition in orthotopic …...pathway may be problematic when it is used...

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