BlockadeofTGF-bSignalingbytheTGFbR-IKinaseInhibitor ... · investigated here the combination effects of radiation and LY2109761 in established human glioblastoma cell lines and in

Microenvironment and Immunology

Blockade of TGF-b Signaling by the TGFbR-I Kinase InhibitorLY2109761 Enhances Radiation Response and ProlongsSurvival in Glioblastoma

Mengxian Zhang1,2,4, Susanne Kleber5, Manuel R€ohrich2,4, Carmen Timke2,4, Na Han1, Jochen Tuettenberg5,Ana Martin-Villalba3, Juergen Debus4, Peter Peschke2, Ute Wirkner2, Michael Lahn6, and Peter E. Huber2,4

AbstractGlioblastoma multiforme (GBM) is a highly aggressive primary brain tumor that tends to be resistant to the

ionizing radiotherapy used to treat it. Because TGF-b is a modifier of radiation responses, we conducted apreclinical study of the antitumor effects of the TGF-b receptor (TGFbR) I kinase inhibitor LY2109761 incombination with radiotherapy. LY2109761 reduced clonogenicity and increased radiosensitivity in GBM celllines and cancer stem–like cells, augmenting the tumor growth delay produced by fractionated radiotherapy in asupra-additive manner in vivo. In an orthotopic intracranial model, LY2109761 significantly reduced tumorgrowth, prolonged survival, and extended the prolongation of survival induced by radiation treatment. Histologicanalyses showed that LY2109761 inhibited tumor invasion promoted by radiation, reduced tumor microvesseldensity, and attenuated mesenchymal transition. Microarray-based gene expression analysis revealed signalingeffects of the combinatorial treatments that supported an interpretation of their basis. Together, these resultsshow that a selective inhibitor of the TGFbR-I kinase can potentiate radiation responses in glioblastoma bycoordinately increasing apoptosis and cancer stem–like cells targeting while blocking DNA damage repair,invasion, mesenchymal transition, and angiogenesis. Our findings offer a sound rationale for positioning TGFbRkinase inhibitors as radiosensitizers to improve the treatment of glioblastoma.Cancer Res; 71(23); 7155–67.�2011AACR.

Introduction

Glioblastoma multiforme (GBM) continues to be the mostfrequent and most malignant human brain tumor, which ishighly resistant to current conventional treatments and hasone of the worst survival rates among all human cancers.New strategies to treat this deadly disease are desperatelyneeded. Emerging evidence suggests that many cancers,including hematopoietic and solid tumors, may be drivenby a small subpopulation of cancer stem–like cells [CSLC; or

cancer stem cells (CSC); or tumor-initiating cells (TIC)].GBMs are among the first solid cancers in which CSLCswere identified (1, 2). CSLCs markers such as CD133 and sidepopulation have been used to prospectively isolate a smallfraction of cells in human brain tumors with increasedpotential to generate tumor neurospheres and xenografts(3, 4). Recent reports suggest that the expression of theCD133 antigen in gliomas and other brain tumors couldserve as a prognostic indicator for tumor recurrence, malig-nant progression, treatment resistance, and patient survival(5). Several studies indicate that conventional radiationtherapies appear to predominantly target the better differ-entiated CD133� population while leaving many CD133þ

CSLCs alive (6). Thus, targeting CSLCs using inhibitors ofTGF-b could be a promising attempt to improve glioblas-toma treatment (7).

TGF-b is a family of polypeptides that regulates a widevariety of biologic functions including cell proliferation,migration, survival, angiogenesis, immunosurveillance, andembryonic stem cell maintenance and differentiation. Themultifunctional effects of TGF-b are elicited through dimer-ization of the type I (TGFbR-I) and type II (TGFbR-II) serine/threonine kinase receptors. Upon TGF-b binding, the recep-tor complex phosphorylates the transcription factorsSMAD2 and SMAD3, which then binds to SMAD4 andtranslocates to the nucleus, where they regulate transcrip-tion of various target genes (8). The overexpression of TGF-b

Authors' Affiliations: 1Department of Oncology, Tongji Hospital, TongjiMedical College, Huazhong University of Science & Technology, Wuhan,China; Departments of 2Radiation Oncology and 3Neurobiology of BrainTumors, German Cancer Research Center (DKFZ); 4Department ofRadiation Oncology, University Hospital Center, Heidelberg; 5SHG-KlinikIdar-Oberstein Klinik f€ur Neurochirurgie, 2 Dr.-Ottmar-Kohler-Straße, Idar-Oberstein, Germany; and 6Oncology Early Clinical Investigation, LillyResearch Laboratories, Indianapolis, Indiana

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

Corresponding Author: Peter E. Huber, Department of Radiation Oncol-ogy, German Cancer Research Center (DKFZ)/University of HeidelbergMedical Center, 280 Im Neuenheimer Feld, Heidelberg 69120, Germany.Phone: 49-6221-42-2515; Fax: 49-6221-42-2514; E-mail:[email protected]

doi: 10.1158/0008-5472.CAN-11-1212

�2011 American Association for Cancer Research.

CancerResearch

www.aacrjournals.org 7155

on June 11, 2020. © 2011 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Published OnlineFirst October 17, 2011; DOI: 10.1158/0008-5472.CAN-11-1212

http://cancerres.aacrjournals.org/

ligands has been reported in various malignant entitiesincluding malignant gliomas (9–11). In patients with malig-nant glioma, elevated levels of TGF-b have been reported tobe associated with high tumor grade, advanced tumorstages, and poor patient outcome (9, 12). In addition to beimplicated in invasion and intratumoral angiogenesis inglioblastoma, TGF-b signaling has also been reported toplay a pivotal role in maintenance of stemness of gliomaCSLCs (13, 14). These multiple roles of TGF-b in gliomainitiation and progression have promoted the developmentof therapeutic agents based on the inhibition of the TGF-bpathway (13, 15). LY2109761, a novel TGFbR-I kinase inhib-itor, has shown a SMAD2-selective inhibitory profile withantitumor activity in various tumor models such as colo-rectal cancer (16), pancreatic cancer (17), and hepatocellularcarcinoma (18).

Because radiotherapy is a mainstay treatment modalityin glioblastoma but may promote radioresistance in gliomastem cells (6), a combination with an inhibitor of TGF-b,which targets in particular CSLCs (7), seems promising. Weinvestigated here the combination effects of radiation andLY2109761 in established human glioblastoma cell lines andin GBM CSLCs in vitro and in vivo in a subcutaneous and anorthotopic tumor model. Our data indicate that LY2109761is an effective treatment approach alone and augmentsthe radiation treatment response in particular in GBMCSLCs.

Materials and Methods

Cell cultures and treatment conditionsThe human GBM cell lines U87MG and T98 were obtained

from the American Type Culture Collection. CD133þ glio-blastoma CSLCs (NMA-23) were maintained in their undif-ferentiated state using neurobasal media supplemented withepidermal growth factor and fibroblastic growth factor,sodium pyruvate, glutamine, B27, nonessential amino acids,and penicillin/streptomycin (Gibco). NMA-23 cells wereisolated from human glioblastoma surgical sample usingfluorescence-activated cell-sorting (FACS) method with aphycoerythrin (PE)-labeled CD133 antibody (Miltenyi Bio-tec) as previously described (19). The collection of humanbiopsy tissue was approved by the regional ethical commit-tee. LY2109761 was kindly provided by Eli Lilly, constitutedin dimethyl sulfoxide (10 mmol/L), and stored at �20�C. Cellexposures with LY2109761 were conducted 2 hours prior toirradiation with 6 MV X-rays (Mevatron, Siemens) at a doserate of 2.5 Gy/min.

Short interfering RNA treatmentValidated Stealth RNAi (Invitrogen) specific to TGFbR-I was

transfected into U87MG and NMA-23 by using LipofectamineRNAiMAX (Invitrogen) according to the manufacturer's pro-tocol. Stealth RNAi negative control with low GC content(Invitrogen) was used as negative control. The expression ofTGbFR1 was measured by quantitative real-time PCR (qRT-PCR), and phosphorylation of SMAD2 was examined usingWestern blot analysis.

Clonogenic assayIncreasing numbers of cells [pretreated with LY2109761

for 2 hours or transfected with short interfering RNA (siRNA)targeting TGFbR-I or nonsense siRNA for 48 hours] wereplated in 25 cm2

flasks, irradiated, and returned to theincubator for 10 to 14 days. Colonies formed were stainedwith crystal violet (Sigma) and those with at least 50 cellswere counted by microscopic inspection. The linear qua-dratic equation was fitted to data sets to generate survivalcurves, and dose enhancement factor for drugs was calcu-lated at 10% surviving fraction (DEF0.1).

Neurosphere formation, limiting dilution, andproliferation assay

For the neurosphere formation assay, 1,000 NMA-23 cells(pretreated with LY2109761 for 2 hours or transfected withsiRNA targeting TGbFR-I or nonsense siRNA for 48 hours) wereseeded on 25 cm2

flasks and irradiated. After 7 days ofincubation, numbers of neurospheres consisting of at least50 cells were counted under a microscope. For the limitingdilution assay, increasing numbers of NMA-23 cells were platedin 96-well plates in 200mL serum-freemediumand treatedwithradiation or LY2109761 or their combination. After 7 days ofculture, the percentage of wells not containing neurospheresfor each cell-plating density was calculated and plotted againstthe number of cells plated per well. For the proliferation assay,50,000 NMA-23 cells were seeded on 25 cm2

flasks overnightand then treated as described, incubated for another 72 hours,when the numbers of living cells were counted.

Immunofluorescent staining for gH2AXCells were grown and treated in chamber slides. At specified

time points, cells were fixed with 3% paraformaldehyde for 10minutes and then permeabilized with 0.5% Triton X-100 for 30minutes on ice. Antibody [AlexaFluor 488 anti-H2AX phos-phorylated (Ser139); Biolegend] was added at a dilution of1:100 in 3% bovine serum albumin and incubated overnight at4�C. Cells were washed and nuclei were counterstained with 1mg/mL 40,6-diamidino-2-phenylindole. Foci were counted bythe automated image analysis system Metacyte with a ZeissAxioplan-2 imaging epifluorescence microscope equippedwith ISIS software (MetaSystems). For each treatment condi-tion, gH2AX foci were analyzed in at least 400 cells andmediannumber of foci was determined.

Apoptosis and flow cytometryTwenty-four hours after treatments, U87MG and NMA-23

cells were prepared for FACS analysis (FACScan; Becton Dick-inson). Cells were fixed in 70% ethanol, centrifuged, washed inPBS, and the supernatant was removed. Cells were resus-pended in the staining solution of PBS, RNase (200 mg/mL),and propidium iodide (10 mg/mL) and were analyzed for cellswith sub-G1 DNA content. To measure caspase-3 activity, cellswere treated according to the manufacturer's suggestions (BDPharmingen), incubated for 20 minutes on ice, pelleted,washed, and resuspended in washing buffer plus PE-conjugat-ed monoclonal active caspase-3 (BD Pharmingen) and ana-lyzed by flow cytometry and FlowJo software (Tree Star, Inc.).

Zhang et al.

Cancer Res; 71(23) December 1, 2011 Cancer Research7156




Western blot analysisSix hours after treatment as indicated, cells were washed

twice with PBS, and lysed using Qproteome MammalianProtein Prep Kit (QIAGEN GmbH) supplemented with a com-plete protease and phosphate inhibitor cocktail (Sigma). Pro-tein concentration was measured using a Bradford assay(Pierce Chemical Co.), and samples were immunoblotted withantibodies against phospho (p)-SMAD2 and total SMAD2 (CellSignaling) with an anti-b-actin monoclonal antibody (Sigma)as internal loading control.

Microarray analysisU87MG cells were solubilized and homogenized in TRIzol

(Invitrogen) 6 hours after radiation with or without LY2109761treatment (4 Gy, 10 mmol/L). For LY2109761 treatment, cellswere exposed to LY2109761 for 2 hours prior to irradiation as inthe other in vitro assays. Total RNA was isolated according tothemanufacturer's instruction, purity and integrity of the RNAwere assessed with Agilent 2100 BioAnalyzer (Agilent Tech-nologies), and 200 ng of total RNA was used for amplificationand labeling using the Low Input Quick Amp Labeling Kit(#5190-0442) from Agilent following the detailed kit protocol.Cyanine 3–labeled cRNA was purified using RNeasy mini spincolumns fromQIAGEN. Quantity and cyanine incorporation oflabeled cRNAs were determined using a Nanodrop ND-1000spectrophotometer (Peqlab Biotechnologies GmbH). Thelabeled probes were then hybridized to an Agilent 4 � 44 Kwhole human genome microarray (G4112F) containing 45,015features representing 41,000 unique probes. After washing,microarrays were scanned using an Agilent Array scanner(Agilent Technologies). Data were extracted with Agilent fea-ture extraction software (Agilent version 9.1) and statisticallyanalyzed with SUMO software as previously described (20).Ingenuity Pathway Analysis was used for functional analysis(IPA; Ingenuity Systems, Inc.; ref. 21). Microarray data weredeposited in "ArrayExpress" (Accession No.: E-TABM-1148).

qRT-PCRTo quantify mRNA expression, qRT-PCR was carried out

using QuantiTect Primer assay (QIAGEN GmbH) and Quanti-Tect SYBR Green RT-PCR Kit (#204243; QIAGEN) on a Light-Cycler 480 instrument (Roche Diagnostics). The relative expres-sion of the target genes was calculated by normalizing the Cp(crossing point) valueswith those of housekeeping geneGAPDH.

Tumor growth in the subcutaneous U87MG model inBALB/c miceAll animal experiments were approved by in-house and

governmental animal protection committees. A total of 5 �106 U87MG cells were injected subcutaneously into the righthind limb of 6- to 8-week-old BALB/c athymic nude mice(Charles River Laboratories). Animals were randomized into4 groups (control, LY2109761, radiation, radiation plusLY2109761; n ¼ 10 each). Treatments started when tumorswere established and reached a volume of approximately 150mm3. Tumors were irradiated with fractionated radiotherapy(5� 2 Gy, days 0–4 for 5 consecutive days) using a 6MV LINAC(Siemens). LY2109761 was administered orally at 50 mg/kg

twice daily (days 1–5 of eachweek) until the end of observation.Tumor volume for the subcutaneous experiment was deter-mined 3 times weekly by direct measurement with calipers(volume ¼ length � width � width � 0.5).

Tumor growth and animal survival in the orthotopicCSLC model in SCID mice

Beige severe-combined immunodeficient (SCID) mice (8-week-old, 20 g; Charles River Laboratories) were anesthetizedand stereotactically inoculated with NMA-23 cells (104 cells in2mL PBS) into the left forebrain (2mm lateral, 1mmanterior tobregma, at 3 mm depth from skull surface). Animals wererandomized into 4 treatment groups (control, LY2109761,radiation, radiation plus LY2109761; n ¼ 13 each, with 4 micescheduled for histology). LY2109761 treatment (50mg/kg twicedaily) started on day 1 after tumor inoculation and was admin-istered 5 days weekly until the end of observation. Radiationwas delivered on day 4 to the entire head of anesthetized mice(7 Gy single dose) using the 6 MV LINAC. On day 15, animalswere examined by MRI at 1.5 T (Siemens Magnetom Vision)using a custom-made small animal solenoid Tx/Rx radiofre-quency coil. Tumor volumeswere estimated using gadolinium-enhanced T1-weighted spin-echo images. A 3-dimensionalreconstruction of the tumor surface and calculation of thetumor volume were conducted with the QuickVol software aspreviously described (22). For survival studies, moribund miceor mice with severe neurologic symptoms were euthanized.

Tumor histology and immunohistochemistryFour animals per group were sacrificed on day 16 and

exsanguinated by transcardial perfusion first with ice-coldPBS. Brains were dissected, partially cryofixated, or embed-ded in paraffin after fixation in 4% paraformaldehyde for 24hours. Sections of paraffin-embedded blocks were stainedwith hematoxylin and eosin. Immunohistochemistry wascarried out with frozen sections as previously described(23). Primary antibodies included the following: anti-vimen-tin (rabbit polyclonal; Abcam; 1:200), anti-CD31 (rabbitpolyclonal; BD Biosciences Pharmingen; 1:100), anti-fibro-nectin (rabbit polyclonal; Abcam; 1:400), anti-COL5A1 (rab-bit polyclonal; Santa Cruz; 1:100), and anti-YKL-40 (rabbitpolyclonal; Quidel; 1:200). After incubation with primaryantibodies, the appropriate fluorescence-labeled secondaryantibodies were applied. Images were captured using aNikon Eclipse E600 microscope equipped with a Nikondigital sight DS-U1 camera and subsequently analyzed usingImageJ software (NIH, Bethesda, MD). For each treatmentcondition, the analysis was conducted in at least 5 randomlychosen fields from 3 to 5 sections.

Statistical analysisThe unpaired 2-tailed t test was used for the comparison of

parameters between groups. The Kaplan–Meier method wasused to determine the median survival time (MST), and a log-rank test was used to compare the differences between survivalcurves. A value of P < 0.05 was considered significant. Thestatistical analysis was conducted using the software packageStatistika 6.0 (Statsoft).

LY2109761 and Radiation in an Orthotopic Glioblastoma Model

www.aacrjournals.org Cancer Res; 71(23) December 1, 2011 7157




Results

LY2109761 enhanced radiosensitivity of glioblastomacells

To determine the effects of LY2109761 on GBM tumor cellradiosensitivity, clonogenic survival analysis was conducted.LY2109761 pretreatment reduced clonogenic survival in cellcultures of U87MG (Fig. 1A) and T98 (Fig. 1B) followingradiation, resulting in an increase in the radiosensitivity witha DEF0.1 of 1.30 and 1.37, respectively. Likewise, TGbFR-IsiRNA reduced TGbFR-I mRNA expression and inhibitedSMAD2 phosphorylation (Supplementary Fig. S1), associatedwith an increase in the radiosensitivity with a DEF0.1 of 1.34in U87MG cells (Fig. 1C), supporting the notion thatLY2109761 exhibits its radiosensitizing effect via TGF-bsignaling blockage.

LY2109761 reduced GBM-derived CSLC self-renewal andproliferation and sensitizes them to radiation

Considering that glioblastoma stem cells have beenlinked to radioresistance, we explored whether LY2109761would inhibit the self-renewal of GBM CSLCs and increasethe sensitivity of these TICs toward radiation. We usedneurosphere formation as a surrogate marker of the clono-genic survival of glioblastoma CSLCs given they grow insuspension (24). We found that LY2109761 (10 mmol/L) orradiation (4 Gy) alone reduced neurosphere-forming effi-ciency in NMA-23 cells. The combination of LY2109761 plusradiation had supra-additive effects in neurosphere forma-tion and limiting dilution assays (Fig. 2A, B, and D).Likewise, blockage of TGF-b signaling via TGbFR-I siRNAalone or combined with radiation also reduced clonogeni-city of NMA-23 cells (Fig. 2C). Furthermore, a proliferationassay revealed a reduction of NMA-23 cells proliferation/viability after LY2109761 or radiation treatment alone, andtheir combination resulted in a further reduction of the cellcount (Fig. 2E).

LY2109761 enhanced radiation-induced DNA damageand apoptosis rates in glioblastoma cells and GBM-derived CSLCs

DNA damage and repair are important componentsof radiation-induced cytotoxicity. As a marker of recognizedDNA double-strand breaks (DSB), we evaluated the induc-tion of nuclear foci of phosphorylated histone H2AX(gH2AX). U87MG and NMA-23 cells were pretreated withLY2109761 (10 mmol/L) for 2 hours and irradiated (2 Gy). Inboth cells, LY2109761 significantly increased the number ofradiation-induced gH2AX foci at 30 minutes and 24 hourspostirradiation (Fig. 3A–C), whereas LY2109761 monother-apy did not significantly alter (P > 0.5) the number of

Radiation dose (Gy)

1086420

Su

rviv

ing

fra

cti

on

0.001

0.01

0.1

1

Control

LY 10 μmol/L

DEF0.1 = 1.30

U87MG

Radiation dose (Gy)

1086420

Su

rviv

ing

fra

cti

on

0.001

0.01

0.1

1

Control

LY 5 μmol/L

DEF0.1 = 1.37

T98

Radiation dose (Gy)

1086420

Su

rviv

ing

fra

cti

on

0.001

0.01

0.1

1

Untreated control

Negtive control

TGFβR1 siRNA

DEF0.1 = 1.34

U87MG

A

B

C

Figure 1. The effects of LY2109761 or TGFbR1 siRNA on radiosensitivityof glioblastomacell linesmeasuredbyclonogenic survival assay.U87MGand T98 cells were pretreated with 10 and 5 mmol/L of LY2109761,respectively (which resulted in a surviving fraction of about 80%, data notshown), 2 hours before irradiation or transfected with siRNA targetingTGbFR-I or negative nonsense siRNA for 48 hours before radiation.Colony-forming efficiency was determined 10 to 14 days later, andsurvival curves were generated after correction for the cytotoxicityinduced by LY2109761 alone and linear quadratic equation was fitted to

data sets. A, U87MG treated with or without LY2109761. B, T98 treatedwith or without LY2109761. C, U87MG treated with siRNA or negativecontrol (DEF0.1 ¼ 1.34 when siRNA vs. negative control). Points, mean;bars, SD; DEF, dose enhancement factor; LY, LY2109761.

Zhang et al.





gH2AX foci. These data suggested that LY2109761increased radiation-induced DSBs and inhibited DNA dam-age repair.Next, we analyzed whether LY2109761 also affected apopto-

sis. We found in both U87MG and NMA-23 cells that a 2-hourpretreatment with LY2109761 before irradiation increasedapoptosis rates in a supra-additive manner in both sub-G1

and caspase-3 activity assays at 24 hours after radiation(Fig. 3D and E).

LY2109761 suppressed SMAD2 phosphorylation in bothglioblastoma cells and GBM-derived CSLCs

Because SMAD proteins are central mediators of signalsfrom TGF-b receptors, we evaluated the effect of LY2109761 onthe phosphorylation of SMAD2 (p-SMAD2), one of their imme-diate downstream targets. As expected, LY2109761 (10 mmol/Lfor U87MG and NMA-23; 5 mmol/L for T98) alone or combi-nation with radiation (4 Gy) effectively suppressed SMAD2phosphorylation (Fig. 4).

Figure 2. LY2109761 or TGFbR1siRNA reduces NMA-23 self-renewal and proliferation andsensitizes them to radiation. A,representative images (�40) ofneurospheres formed by NMA-23cells treated with LY2109761 (LY;10 mmol/L) or radiation (RT; 4 Gy) ortheir combination. B, the number ofneurospheres per flask, comparedbetween the control group and cellstreated as indicated. C, the numberof neurospheres per flask,compared between the untreatedgroup and cells treated withnegative control, siRNA, RT, orsiRNA þ RT. D, NMA-23 cell self-renewal capacity was evaluated in alimiting dilution assay under eachtreatment condition. The data arepresented as the percentage ofwells not containing neurospheresfor each cell-plating density. E,NMA-23 cell proliferation wasdetermined by cell count after 72hours of exposure to eachtreatment. Relative numbers of cellsare shown as histogram. Columns,mean; bars, SD; �, P < 0.05 versuscontrol (or negative control);��, P < 0.05 versus control(or negative control) and eachmonotherapy; #, P > 0.05 versusuntreated.

A LYControl

LY + RTRT

LY + RTRTLYControl

Nu

mb

er

of

sp

he

res

0

100

200

300

400

*

*

**

0

10

20

30

40

50

60

70

80

90

100

100806040200% w

ells w

ith

ou

t n

eu

rosp

here

s

Number of cells per well

Control LY RT LY + RT LY + RTRTLYControl

Re

lati

ve

pro

life

rati

on

0

20

40

60

80

100

120

*

*

**

Untr

eate

d

Neg

ative c

ontrol

siRNA

RT

siRNA

+ R

T

Nu

mb

er

of

sp

here

s

0

50

100

150

200

250

300

350

**

**

#

CB

D

E






LY2109761 altered gene expression andpathway analysisin glioblastoma cells

To further investigate the potential molecular basis of theinteraction of LY2109761 and radiation (RT), gene expres-sion analysis was conducted on U87MG cells using micro-arrays. A heatmap was generated (Fig. 5A) representing 988transcripts comparing significantly differentially regulatedgenes for each treatment condition (LY, RT, LY þ RT) versus

untreated controls. Selected genes were found similarlyregulated in NMA-23 cells as in U87MG cells, which wasconfirmed using qRT-PCR (Fig. 5B; Supplementary Fig. S2),including ID1, HEY1, ANGPT2, BIRC2 (downregulated byLY2109761 treatment) and RHOB, LRIG1, ACTG2 (upregu-lated by LY2109761 treatment) as well as AKT2, ATF5, LMO2(upregulated by radiation and reversed by LY2109761pretreatment).

A

H2AX foci number

5452484644423836343228262422181614128642 50403020100

Fre

qu

en

cy o

f cells

0

20

40

60

80

100

Accu

mu

late

d f

req

uen

cy o

f cells (

%)

0

20

40

60

80

100

Control-24 h

LY-24 h

RT-24 h

LY + RT-24 h

Control-24 h

LY-24 h

RT-24 h

LY + RT-24 h

B

Time after radiation

24 h30 min

Med

ian

nu

mb

er

of

foci

0

10

20

30

40 Control

LY

RT

LY + RT

U87MG*

*

Time after radiation

24 h30 min

Med

ian

nu

mb

er

of

foc

i

0

10

20

30

40 Control

LY

RT

LY + RT

NMA-23

*

*

C

Figure 3. LY2109761 increasesradiation-induced gH2AX foci andincreases radiation-inducedapoptosis in U87MG and NMA-23cells. A, representative images ofgH2AX foci immunofluorescentstaining obtained from control andtreated cells at 24 hours afterradiation in U87MG. B,representative results of the focinumber distribution using anautomated image analysis systemfor treated and control cells at 24hours after radiation in U87MG. Thehistogram depicts the frequency ofthe cells with a specific number offoci and the line scatter plot showsthe accumulated percentage of cellfrequency. C, themedian number offoci compared between controlsand treated groups in U87MG (left)and NMA-23 (right). Columns,mean; bars, SD; �, P < 0.05.

Zhang et al.





Further analysis using IPA of differentially regulated geneswith at least 2-fold change (LY, 111 genes; RT, 36 genes; LY þRT, 157 genes) revealed 8 functional categories with highlyaffected and enriched transcripts (Fig. 5C). The associatedmolecular and cellular functions are related to cellular move-ment, cellular growth and proliferation, gene expression, celldeath, cell cycle, cell-to-cell signaling and interaction, cellsignaling, and molecular transport. For example, the topfunction for LY2109761 and radiation alone or their combina-tion was associated with cellular movement, cell death, andcellular growth and proliferation.Venn diagrams revealed the treatment-specific upregulated

and downregulated genes (Fig. 5D) and also the commonlyaffected genes (Fig. 5D). A significant number of genes werecommonly affected under the treatment of LY2109761 andradiation plus LY2109761, whereas considerably fewer geneswere commonly affected by radiation and radiation plusLY2109761, suggesting a comprehensive impact of LY2109761on radiation response at the gene expression level whencombined with radiotherapy.

LY2109761 enhanced radiation-induced tumor growthdelay in a U87MG subcutaneous xenograft tumor modelin BALB/c nude miceA tumor growth delay experiment was carried out using

U87MG tumors growing subcutaneously in the hind leg of nudemice. As shown in Fig. 6A, the time for 5-fold increase in tumorsize (from 150 mm3 to 750 mm3) was calculated using thetumor volumes from the individual mice in each group. Themean time for 5-fold increase in tumor size increased from 11.4days for sham-treated mice to 17.2 days for LY2109761-treatedmice and 23.9 days for irradiated mice. Radiation plus

LY2109761 increased the time to 36.4 days. The tumor growthdelay versus controls was 5.8 days for LY2109761 alone, 12.5days for radiation alone, and 25.0 days for the combination,indicating a supra-additive effect between LY2109761 andradiation on growth delay of U87MG xenografts.

LY2109761 increased survival in an orthotopical CSLCglioblastoma model and enhanced antitumor activity ofradiation

The effect of LY2109761 with or without radiation onintracranial CSLC (NMA-23) tumor growth was monitored bynoninvasive MRI. By day 15, both LY2109761 and radiation

Figure 3. (Continued ) D,combined treatment withLY2109761 and irradiationsubstantially enhanced apoptosisrates in a supra-additive manner.The percentage of apoptotic cellswas determined by flow cytometricanalysis of cells with sub-G1 DNAcontent and active caspase-3 at 24hours after treatments in U87MG(left) and NMA-23 (right). U87MGcells and NMA-23 cells werepretreated with LY2109761(10 mmol/L) for 2 hours andirradiated (8 Gy). Columns, mean;bars, SD; �, P < 0.05 versus control;��, P < 0.05 versus control andrespective single treatment. E,representative results of flowcytometric analysis for activecaspase-3 using FlowJo softwareat 24 hours after treatments.

LY + RTRTLYControl

0

100 101 102 103 104

Co

un

t

FL1-H

Sample name

IV + radiation–1.010radiation–1.007Iv–1.004Ctrl–1.001

Ap

op

toti

c c

ell

s (

%)

0

5

10

15

20Active caspase-3

Sub-G1

** *

*

**

**U87MG

LY + RTRTLYControl

Ap

op

toti

c f

racti

on

(%

)

0

5

10

15

20Active caspase-3

Sub-G1

NMA-23

** *

*

** **

D

E

LY2109761

Radiation

p-SMAD2

Total SMAD2

ββ-Actin

— + — +

—— + +

— + — + — + — +

—— + + —— + +

NMA-23T98U87MG

Figure 4. The effects of LY2109761 on the activation of SMAD2-dependent downstream pathway of TGF-b in U87MG, T98, and NMA-23cells. Cells were treated with LY2109761 (10 mmol/L for U87MG andNMA-23; 5 mmol/L for T98), or radiation (4 Gy), or their combination; 6hours later, cells were lysed and whole-cell lysates were subjected toWestern blot analysis with anti-phospho-SMAD2 and anti-total SMAD2antibodies. b-Actin monoclonal antibody was used as an internal loadingcontrol.






monotherapy markedly inhibited tumor growth by 43.4% and54.3% versus controls (Fig. 6B and C), which was furtherreduced by the combination to 76.3%. The Kaplan–Meiersurvival curves showed that LY2109761 alone increased mod-

estly but statistically significantly animal survival comparedwith controls, with a MST of 20 versus 18 days (P < 0.05, log-rank test; Fig. 6D); radiotherapy alone prolonged theMST to 22days. Importantly, the combination treatment with LY2109761

Time hold

BA

C

Cellular movement

LY vs. DMSO-filtered - 2010-10-24 RT vs. DMSO-filtered - 2010-10-24 LY plus RT vs. DMSO-filtered - 2010-10-24

Cellular growth and proliferation

Gene expression

Cell death

Cell cycle

Cell-to-cell signaling and interaction

Cell signaling

Molecular transport

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0

AKT2–1.52

–2.11

ID1

–11.09

–9.71

ANGPT2

–2.97

–2.19

BIRC2

–2.31

–1.71

RHOB

3.04

3.89

LRIG1

2.21

2.93

ACTG2

4.51

5.48

HEY1

–1.92

–2.56

Microarray

qRT-PCR

Microarray

qRT-PCR

0

–0.5

–1

–1.5

–2

–2.5

–3

–3.5

8

6

4

2

0

–2

–4

–6

–8

–10

–12

< = –100 > = 1000.00

6h

–D

MS

O 1

6h

–D

MS

O 2

6h

–D

MS

O 3

6h

–R

T 1

6h

–R

T 2

6h

–R

T 3

6h

–L

y 1

6h

–L

y 2

6h

–L

y 3

6h

–L

y +

RT

1

6h

–L

y +

RT

2

6h

–L

y +

RT

3

Fold

expre

ssio

n

(RT

+ L

Y v

s. R

T)

Fold

expre

ssio

n

(LY

vs. contr

ol)

AKF5–1.52

–1.61

LMO2–2.87

–2.54

4.5

-log (P)

5.0 6.0 7.0 8.0 9.05.5 6.5 7.5 8.5 9.5

@2000-2010 Ingenuity Systems, Inc. All rights reserved.

Figure 5. The effects of eachtreatment on gene expressionprofiles generated from U87MGcells. A, a heatmap representing988 transcripts was generatedusing 4-class t test (P ¼ 1.0E-004).Sets of differentially regulatedgenes from 3 biologic replicateswere identified for each treatmentcondition (LY, P ¼ 1.0E-004; RT, P¼ 2.0E-003; LYþRT,P¼ 1.0E-003)comparedwith the gene expressionprofile from untreated control using2-class t test. Color intensity isassigned to ratios of geneexpression; shades of red, genesthat are upregulated; shades ofgreen, genes that aredownregulated; black, genes thatare unchanged. B, fold changes inexpression levels of genesregulated by LY2109761 (top) andthose upregulated by radiation andreversed by radiation plusLY2109761 (bottom) as determinedby microarray and qRT-PCRmethods. C, bar graph representingfunctions significantly differentbetween treatments from IPAderived from genes with more than2-fold change. X-axis indicated thesignificant score in each functionalcategory. Given the degree ofenrichment in the functions, whichis reflected by the significancescore (negative logarithm of the Pvalue), the results indicate that thegenes affected by treatments donot simply comprise a random listand may therefore be of potentialfunctional relevance.

Zhang et al.





plus radiotherapy further increased the MST to 28 days(P < 0.05 vs. respective monotherapies) indicating a supra-additive effect on tumor growth and animal survival betweenLY2109761 and radiotherapy in the orthotopic glioblastomamodel.

LY2109761 inhibited tumor invasion and reduced tumormicrovessel density

Previous studies have shown that glioblastoma cells canexpress the intermediate neurofilament vimentin, includingthose tumor cells that infiltrate into normal parenchyma. Toevaluate tumor invasiveness in intracranial tumor sections,hematoxylin–eosin and immunohistochemical staining forvimentin was done. As shown in Fig. 7A and B, sham-treatedintracranial xenografts displayed peripheral invasion of thesurrounding brain as single cells and cell clusters, frequentlysurrounded by numerous small satellite tumors. Interest-ingly, a more invasive growth pattern was observed afterradiotherapy alone than controls. In contrast, tumors frommice receiving LY2109761 monotherapy or LY2109761 plusradiotherapy displayed a significantly reduced invasiongrowth pattern compared with the control and radiotherapygroup.

Next, we investigated tumor angiogenesis as anotherimportant process of tumor growth particularly pertinentin glioblastoma using CD31 endothelial staining and micro-vessel density (MVD) analysis. We found that LY2109761alone (MVD: 39.2 � 8.4) and radiotherapy alone (47.7 � 10.5)

D Up regulated

73

61

93

8

5

6151 96

4

448

112

120

LV RT (179)LV RT (167)

LY (145) LY (155)RT (170) RT (127)

7

Down regulated

Figure 5. (Continued ) D, Venn diagrams depicting the number of genesthat were commonly upregulated or downregulated by differenttreatments. DMSO, dimethyl sulfoxide.

A

Days after treatment start

403020100

Tu

mo

r vo

lum

e (

mm

3)

Tu

mo

r vo

lum

e (

mm

3)

0

500

1,000

1,500

2,000

2,500

ControlRTLY + RTLY + RT

*

*

**

LYControl

LY + RTRT

B

LY + RTRTLYControl

0

20

40

60

80

100

120

140

160

180

@@

@@

Time (d)

30252015

Su

rviv

al

0.0

0.2

0.4

0.6

0.8

1.0

Control

LY

RT

LY + RT

* *

DC

Figure 6. In vivo antitumor activity of LY2109761 with or without radiation in a subcutaneous model and in an independent orthotopic glioblastoma model. A,BALB/c nu/nu mice with U87MG human glioblastoma growing subcutaneously were treated as described in Materials and Methods. Tumor volumeswere calculated using the formula: v¼ length�width�width� 0.5. Data,means�SE; n¼ 10 animals in each group. Statistical significance is designated as:�, P < 0.05 versus control; ��, P < 0.05 versus control and each monotherapy. B, representative gadolinium-enhanced T1-weighed images of orthotopicxenograft from sham-treated control animals and animals that had received LY2109761 (LY) or radiation (RT) or their combination examined by MRI 15 daysafter tumor injection. C, tumor volumes estimated fromMRI. @,P < 0.05 versus control; @@, P < 0.05 versus control and eachmonotherapy. D, Kaplan–Meiersurvival analysis of beige SCID mice transplanted intracranially with NMA-23 cells treated with LY2109761 or radiation or their combination. Differencesbetween survival curves were compared using a log-rank test. �, P < 0.05 for MST.






reduced MVD versus controls (62.2 � 10.55, P < 0.05; Fig. 7B)More importantly, LY2109761 plus radiation resulted in afurther reduction in MVD (23.4 � 5.9) versus monotherapies(P < 0.05), suggesting a beneficial combination effect onreducing tumor angiogenesis in the orthotopic CSLC model(Fig. 7B and C).

LY2109761 inhibited mesenchymal change in the CSLCorthotopic glioblastoma model

A mesenchymal character has been associated withincreased malignancy, treatment resistance, and poor prog-nosis in glioblastoma. We found here that LY2109761 inhibitedthe mesenchymal character in our orthotopic CSLCs model.Immunohistochemical staining showed that LY2109761 aloneand in combination with radiotherapy reduced the expressionof established mesenchymal marker proteins including fibro-nectin, COL5A1, and YKL-40 (refs. 23, 25; Fig. 7B).

Discussion

The pivotal role of TGF-b in promoting cellular processesthat are important for glioblastoma progression suggests thatthis pathway may be a promising target for therapy. In thepresent study, we provide evidence that TGF-b signalingblockade by the small-molecule TGFbR-I kinase inhibitor,LY2109761, is an effective treatment approach and can more-over augment radiation response in human glioblastoma.This conclusion is supported by several pieces of evidence.First, LY2109761 significantly enhanced radiation-inducedcytotoxicity and cooperated with radiation to suppress clo-nogenic survival of established glioblastoma cell lines. Mech-anistic studies revealed that LY2109761 may act in concertwith radiation to enhance radiation-induced DNA damageand apoptosis. Second, LY2109761 also increased the sensi-tivity of primary GBMCSLCs derived from surgical specimenstoward radiation. Third, LY2109761 enhanced radiation-induced tumor growth delay in 2 independent models: inU87MG tumors growing subcutaneously in BALB/c nu/numice and in orthotopic CSLC tumors growing in brains ofSCID mice. In this orthotopic brain tumor model, LY2109761increased animal survival alone and increased the radiation-induced prolongation of the animal life span.

The interesting finding that TGF-b inhibition impedes thecellular DNA damage stress response and results in increasedradiosensitivity had been shown in mammary epithelial cells(26). While these authors reported a reduced gH2AX focinumber following TGF-b inhibition, we found here an increaseof radiation-induced gH2AX foci number that may be due tothe difference in cell types or TGF-b inhibitors used.

Targeting CSLCs in human tumors including glioblastoma isan evolving concept for developing new treatment options incancer. Stem cells have been reported to be responsible for theinitiation, propagation, recurrence, and radioresistance of glio-mas (6). Notably, themost effective targeted therapiesmay haveactivity against CSLCs. Encouragingly, we found that LY2109761reduced the self-renewal and proliferation capability of GBMCSLCs and also enhanced the radiosensitivity of GBM CSLCs.More importantly, LY2109761 further increased the antitumoreffects of radiation and increased animal survival when GBMCSLCs were injected orthotopically into mice brains.

Invasion is another important feature of human glioblasto-ma responsible for their dismal outcomes. Although ionizingradiation is the mainstay of nonsurgical treatment in GBM,radiation may also promote migration and invasiveness ofglioma cells (27), which has been attributed, in part, to

A YLControl

RT

LYcontrol

LY+RTRT

TB

T

B

T

B

T

B

LY + RT

LY + RT

RTLYControl

Vimentin

CD31

Fibronectin

COL5A1

YKL-40

LY + RTRTLYControl

CD

31

+ p

er

fie

ld (

MV

D)

100

80

60

40

20

0

*

*

**

C

B

Figure 7. Histologic studies of glioblastoma orthotopic xenograftsfrom mice treated with LY2109761 (LY), or radiation (RT), or both.Original magnification, �200. A, representative examples of thehematoxylin–eosin staining. B, representative examples of theimmunofluorescence staining of human vimentin, CD31, fibronectin,COL5A1, and YKL-40. T, tumor; B, normal brain. C, quantitativeanalysis of CD31 staining in tumors given as MVD. For quantificationof MVD, vessels on each section were counted in 5 high-powerfields (magnification, �200). Columns, mean; bars, SD. �, P < 0.05versus control; ��, P < 0.05 versus control and respective singletreatment.

Zhang et al.





increased levels of TGF-b (28). Accordingly, our results indi-cated that radiation may promote glioblastoma cell invasion,whereas the blockade of TGF-b signaling using LY2109761inhibited both constitutive and radiation-provoked tumor cellinvasion in the orthotopic model.Activated tumor angiogenesis is another characteristic fea-

ture of GBM, contributing to tumor invasiveness and radio-resistance. The antiangiogenic effects of targeting TGF-b (29,30) could be corroborated in our study because LY2109761alone and in combination with radiation reduced tumorMVD,indicating that LY2109761 has direct antimigratory and anti-angiogenic properties, which can be beneficial in the context ofan attenuation of unwanted side effects of radiotherapy such ascertain promigratory and proangiogenic effects.The fundamental role for mesenchymal change in promot-

ing invasion, treatment response, and even CSC function inhuman carcinoma and GBM invasion is increasingly recog-nized (6, 31). Recent studies have established that TGF-b is amaster regulator of epithelial-to-mesenchymal transition(EMT) in carcinoma (32). To our best knowledge, its relevancetomesenchymal change inGBMmodels has not been reported.Here, we found that the blockade of TGF-b signaling usingLY2109761 markedly reduced the expression of mesenchymalmarkers in the orthotopic glioblastomamodel. It is conceivablethat this inhibitory effect on mesenchymal change ofLY2109761, at least partly, contributes to its antimigratorycapacity.Considering that TGF-b is involved in multiple signaling

pathways, genome-widemicroarray analysis was conducted toinvestigate potential mechanisms of LY2109761, TGF-b signal-ing, and radiation interactions. The regulated genes representfunctional classes involved in diverse biologic processesincluding cellularmovement, cellular growth andproliferation,cell death, cell cycle, cell-to-cell signaling and interaction, andcell signaling. ID1 (along with other ID family members ID2,ID3, and ID4; data not shown), HEY1, ANGPT2, BIRC2 (cIAP1;inhibitor of apoptosis protein 1) were among the genes down-regulated by LY2109761. ID1 has been reported to promoteinvasion, to mediate tumor angiogenesis by production ofVEGF, to be involved in the resistance of cancer cells againstcytotoxic drugs (33), and has also been implicated in CSCfunction (34) as well as EMT process (35). Our data are here inline with a very recent report focusing on the effects ofLY2109761 on ID1 (36). Angiopoietins (ANGPT) are ligands ofthe endothelial cell receptor TIE2 and have crucial roles in thetumor angiogenic switch, inflammation, metastasis, and lym-phangiogenesis. Increased expression of ANGPT2 and higherANGPT2/ANGPT1 ratios in tumors correlate with poor prog-nosis in many cancers. Agents specifically targeting ANGPT1and ANGPT2 are currently in phase II clinical trials (37). Highexpression levels of IAPs that inhibit apoptosis have been

associated with poor treatment response and prognosis (38).In addition, we also identified and confirmed several genesupregulated by LY2109761 treatment, including RHOB, LRIG1,and ACTG2, which act as tumor suppressors and have putativeroles in tumor therapy resistance and DNA damage response(39–41).

Radiotherapy is an effective treatment modality for glio-blastoma. To evade radiation toxicity, certain pathways mightbe activated that contribute to tumor invasiveness, angiogen-esis, and radioresistance (42, 43). The set of such genes thatwere upregulated by radiation and reversed by LY2109761further included AKT, ATF5, and LMO2. AKT is a majordownstream effector of the phosphoinositide 3-kinase (PI3K)pathway, a potent prosurvival pathway, associated with radio-resistance of cancer cells with AKT inhibition having beenshown to increase radiation effects on tumors (44). Activatingtranscription factor 5 (ATF5) is highly expressed in malignantglioma and plays an important role in promoting cell survivalby stimulating transcription of MCL1, an antiapoptotic BCL2family member (45). ATF5 has been shown to be a potentrepressor of p53, and elevated expression of ATF5 in tumor isrelated to radioresistance and greater cell motility (46). TheLIM-domain protein LMO2 is a specific regulator of tumorangiogenesis, which has been suggested an attractive drugtarget in cancer (47). Together these array data may provideadditional molecular signaling hints for the beneficial effectsobserved after combined TGF-b signaling inhibition withLY2109761 and radiation in the treatment of glioblastoma.

In summary, we have provided evidence that LY2109761, aspecific TGFbR-I kinase inhibitor, is active against glioblasto-ma alone and enhances the antitumor efficacy of radiationboth in vitro and in vivo, in particular in GBM CSLCs. Ourfindings rationalize further translational studies of LY2109761or agents with similar properties alone and in combinationwith radiotherapy in the treatment of glioblastoma.

Disclosure of Potential Conflicts of Interest

Michael Lahn is an employee of Lilly, Inc.

Grant Support

This work was supported, in part, by grants from DFG National PriorityResearch Program the Tumor-Vessel Interface (SPP1190), National Aeronauticsand Space Administration Specialized Center of Research NNJ04HJ12G, Kom-petenzverbund Strahlenforschung (KVSF, 03NUK004A,C) of Bundesministerienfuer Bildung, Forschung und Umwelt (BMBF/BMU).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicate thisfact.

Received April 12, 2011; revised August 17, 2011; accepted September 8, 2011;published OnlineFirst October 17, 2011.

References1. Reya T, Morrison SJ, ClarkeMF,Weissman IL. Stem cells, cancer, and

cancer stem cells. Nature 2001;414:105–11.2. Singh SK, Hawkins C, Clarke ID, Squire JA, Bayani J, Hide T, et al.

Identification of human brain tumour initiating cells. Nature 2004;432:396–401.

3. Taylor MD, Poppleton H, Fuller C, Su X, Liu Y, Jensen P, et al. Radialglia cells are candidate stem cells of ependymoma. Cancer Cell2005;8:323–35.

4. Galli R, Binda E, Orfanelli U, Cipelletti B, Gritti A, De Vitis S, et al.Isolation and characterization of tumorigenic, stem-like neural






precursors from human glioblastoma. Cancer Res 2004;64:7011–21.

5. Zeppernick F, Ahmadi R, Campos B, Dictus C, Helmke BM, Becker N,et al. Stem cell marker CD133 affects clinical outcome in gliomapatients. Clin Cancer Res 2008;14:123–9.

6. BaoS,WuQ,McLendonRE,HaoY,ShiQ,HjelmelandAB, et al. Gliomastem cells promote radioresistance by preferential activation of theDNA damage response. Nature 2006;444:756–60.

7. Chinnaiyan P, Huang S, Vallabhaneni G, Armstrong E, Varambally S,Tomlins SA, et al. Mechanisms of enhanced radiation response fol-lowing epidermal growth factor receptor signaling inhibition by erlo-tinib (Tarceva). Cancer Res 2005;65:3328–35.

8. DerynckR, ZhangYE.Smad-dependent andSmad-independent path-ways in TGF-beta family signalling. Nature 2003;425:577–84.

9. Kjellman C, Olofsson SP, Hansson O, Von Schantz T, Lindvall M,Nilsson I, et al. Expression of TGF-beta isoforms, TGF-beta receptors,and SMADmolecules at different stages of humanglioma. Int JCancer2000;89:251–8.

10. von Bernstorff W, VossM, Freichel S, Schmid A, Vogel I, J€ohnkC, et al.Systemic and local immunosuppression in pancreatic cancer patients.Clin Cancer Res 2001;7:925s–32s.

11. Hawinkels LJ, Verspaget HW, van der Reijden JJ, van der Zon JM,Verheijen JH, Hommes DW, et al. Active TGF-beta1 correlates withmyofibroblasts and malignancy in the colorectal adenoma-carcinomasequence. Cancer Sci 2009;100:663–70.

12. Bruna A, Darken RS, Rojo F, Oca~na A, Pe~nuelas S, Arias A, et al. HighTGFbeta-Smad activity confers poor prognosis in glioma patients andpromotes cell proliferation depending on themethylation of the PDGF-B gene. Cancer Cell 2007;11:147–60.

13. Ikushima H, Todo T, Ino Y, Takahashi M, Miyazawa K, Miyazono K.Autocrine TGF-beta signaling maintains tumorigenicity of glioma-ini-tiating cells through Sry-related HMG-box factors. Cell Stem Cell2009;5:504–14.

14. Pe~nuelas S, Anido J, Prieto-S�anchez RM, Folch G, Barba I, CuartasI, et al. TGF-beta increases glioma-initiating cell self-renewalthrough the induction of LIF in human glioblastoma. Cancer Cell2009;15:315–27.

15. UhlM,AulwurmS,Wischhusen J,WeilerM,MaJY,AlmirezR, et al. SD-208, a novel transforming growth factor beta receptor I kinase inhibitor,inhibits growth and invasiveness and enhances immunogenicity ofmurine and human glioma cells in vitro and in vivo. Cancer Res2004;64:7954–61.

16. ZhangB,HalderSK, ZhangS,DattaPK. Targeting transforminggrowthfactor-beta signaling in liver metastasis of colon cancer. Cancer Lett2009;277:114–20.

17. Melisi D, Ishiyama S, Sclabas GM, Fleming JB, Xia Q, Tortora G, et al.LY2109761, a novel transforming growth factor beta receptor type Iand type II dual inhibitor, as a therapeutic approach to suppressingpancreatic cancer metastasis. Mol Cancer Ther 2008;7:829–40.

18. FransveaE,Angelotti U, Antonaci S,GiannelliG.Blocking transforminggrowth factor-beta up-regulates E-cadherin and reduces migrationand invasion of hepatocellular carcinoma cells. Hepatology2008;47:1557–66.

19. CapperD,Gaiser T,HartmannC,Habel A,MuellerW,Herold-MendeC,et al. Stem-cell-like glioma cells are resistant to TRAIL/Apo2L andexhibit down-regulation of caspase-8 by promoter methylation. ActaNeuropathol 2009;117:445–56.

20. Wagner-Ecker M, Schwager C, Wirkner U, Abdollahi A, Huber PE.MicroRNA expression after ionizing radiation in human endothelialcells. Radiat Oncol 2010;5:25.

21. Kumaraswamy S, Chinnaiyan P, ShankavaramUT, Lu X, CamphausenK, Tofilon PJ. Radiation-induced gene translation profiles reveal tumortype and cancer-specific components. Cancer Res 2008;68:3819–26.

22. Schmidt KF, Ziu M, Schmidt NO, Vaghasia P, Cargioli TG, Doshi S,et al. Volume reconstruction techniques improve the correlationbetween histological and in vivo tumor volume measurements inmouse models of human gliomas. J Neurooncol 2004;68:207–15.

23. CarroMS, LimWK, AlvarezMJ, Bollo RJ, Zhao X, Snyder EY, et al. Thetranscriptional network for mesenchymal transformation of braintumours. Nature 2010;463:318–25.

24. Wang J, Wakeman TP, Lathia JD, Hjelmeland AB,Wang XF, White RR,et al. Notch promotes radioresistance of glioma stem cells. StemCells2010;28:17–28.

25. Tso CL, Shintaku P, Chen J, Liu Q, Liu J, Chen Z, et al. Primaryglioblastomas express mesenchymal stem-like properties. Mol Can-cer Res 2006;4:607–19.

26. Kirshner J, Jobling MF, Pajares MJ, Ravani SA, Glick AB, Lavin MJ,et al. Inhibition of transforming growth factor-beta1 signaling attenu-ates ataxia telangiectasia mutated activity in response to genotoxicstress. Cancer Res 2006;66:10861–9.

27. Wild-Bode C, Weller M, Rimner A, Dichgans J, Wick W. Sublethalirradiation promotes migration and invasiveness of glioma cells: impli-cations for radiotherapy of human glioblastoma. Cancer Res2001;61:2744–50.

28. Biswas S, Guix M, Rinehart C, Dugger TC, Chytil A, Moses HL, et al.Inhibition of TGF-beta with neutralizing antibodies prevents radiation-induced acceleration of metastatic cancer progression. J Clin Invest2007;117:1305–13.

29. Koochekpour S, Merzak A, Pilkington GJ. Vascular endothelial growthfactor production is stimulatedbygangliosides andTGF-beta isoformsin human glioma cells in vitro. Cancer Lett 1996;102:209–15.

30. Platten M, Wick W, Wild-Bode C, Aulwurm S, Dichgans J, Weller M.Transforming growth factors beta(1) (TGF-beta(1)) and TGF-beta(2)promote glioma cell migration via Up-regulation of alpha(V)beta(3)integrin expression. Biochem Biophys Res Commun 2000;268:607–11.

31. Phillips HS, Kharbanda S, Chen R, Forrest WF, Soriano RH, Wu TD,et al. Molecular subclasses of high-grade glioma predict prognosis,delineate a pattern of disease progression, and resemble stages inneurogenesis. Cancer Cell 2006;9:157–73.

32. Wendt MK, Allington TM, Schiemann WP. Mechanisms of the epithe-lial-mesenchymal transition by TGF-beta. Future Oncol 2009;5:1145–68.

33. Gautschi O, Tepper CG, Purnell PR, Izumiya Y, Evans CP, Green TP,et al. Regulation of Id1 expression by SRC: implications for targeting ofthe bone morphogenetic protein pathway in cancer. Cancer Res2008;68:2250–8.

34. Tang B, Yoo N, Vu M, Mamura M, Nam JS, Ooshima A, et al. Trans-forming growth factor-beta can suppress tumorigenesis througheffects on the putative cancer stem or early progenitor cell andcommitted progeny in a breast cancer xenograft model. Cancer Res2007;67:8643–52.

35. Zhang X, Ling MT, Wang Q, Lau CK, Leung SC, Lee TK, et al.Identification of a novel inhibitor of differentiation-1 (ID-1) bindingpartner, caveolin-1, and its role in epithelial-mesenchymal transitionand resistance to apoptosis in prostate cancer cells. J Biol Chem2007;282:33284–94.

36. Anido J, S�aez-Border��as A, Gonz�alez-Junc�a A, Rod�on L, Folch G,Carmona MA, et al. TGF-beta receptor inhibitors target the CD44/Id1glioma-initiating cell population in human glioblastoma. Cancer Cell2010;18:655–68.

37. Huang H, Bhat A, Woodnutt G, Lappe R. Targeting the ANGPT-TIE2pathway in malignancy. Nat Rev Cancer 2010;10:575–85.

38. Fulda S. Inhibitor of apoptosis proteins as targets for anticancertherapy. Expert Rev Anticancer Ther 2007;7:1255–64.

39. Prendergast GC. Actin' up: RhoB in cancer and apoptosis. Nat RevCancer 2001;1:162–8.

40. Hedman H, Henriksson R. LRIG inhibitors of growth factor signalling -double-edged swords in human cancer? Eur J Cancer 2007;43:676–82.

41. Watson MB, Lind MJ, Smith L, Drew PJ, Cawkwell L. Expressionmicroarray analysis reveals genes associatedwith in vitro resistance tocisplatin in a cell line model. Acta Oncol 2007;46:651–8.

42. Abdollahi A, Lipson KE, Han X, Krempien R, Trinh T, Weber KJ, et al.SU5416 and SU6668 attenuate the angiogenic effects of radiation-induced tumor cell growth factorproductionandamplify thedirect anti-endothelial action of radiation in vitro. Cancer Res 2003;63:3755–63.

43. Timke C, Zieher H, Roth A, Hauser K, Lipson KE, Weber KJ, et al.Combination of vascular endothelial growth factor receptor/platelet-derived growth factor receptor inhibition markedly improves radiationtumor therapy. Clin Cancer Res 2008;14:2210–9.

Zhang et al.





44. Bussink J, van der Kogel AJ, Kaanders JH. Activation of the PI3-K/AKTpathway and implications for radioresistancemechanisms in head andneck cancer. Lancet Oncol 2008;9:288–96.

45. Sheng Z, Li L, Zhu LJ, Smith TW, Demers A, Ross AH, et al. A genome-wide RNA interference screen reveals an essential CREB3L2-ATF5-MCL1 survival pathway in malignant glioma with therapeutic implica-tions. Nat Med 2010;16:671–7.

46. Nishioka T, Miyai Y, Haga H, Kawabata K, Shirato H, Homma A, et al.Novel function of transcription factor ATF5: blockade of p53-depen-dent apoptosis induced by ionizing irradiation. Cell Struct Funct2009;34:17–22.

47. Yamada Y, Pannell R, Forster A, Rabbitts TH. The LIM-domain proteinLmo2 is a key regulator of tumour angiogenesis: a new anti-angio-genesis drug target. Oncogene 2002;21:1309–15.






2011;71:7155-7167. Published OnlineFirst October 17, 2011.Cancer Res Mengxian Zhang, Susanne Kleber, Manuel Röhrich, et al. Glioblastoma

inLY2109761 Enhances Radiation Response and Prolongs Survival R-I Kinase Inhibitorβ Signaling by the TGFβBlockade of TGF-

Updated version

10.1158/0008-5472.CAN-11-1212doi:

Access the most recent version of this article at:

Material

Supplementary

http://cancerres.aacrjournals.org/content/suppl/2011/10/17/0008-5472.CAN-11-1212.DC1

Access the most recent supplemental material at:

Cited articles

http://cancerres.aacrjournals.org/content/71/23/7155.full#ref-list-1

This article cites 47 articles, 15 of which you can access for free at:

Citing articles

http://cancerres.aacrjournals.org/content/71/23/7155.full#related-urls

This article has been cited by 21 HighWire-hosted articles. Access the articles at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected]

To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at

Permissions

Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://cancerres.aacrjournals.org/content/71/23/7155To request permission to re-use all or part of this article, use this link



http://cancerres.aacrjournals.org/lookup/doi/10.1158/0008-5472.CAN-11-1212

http://cancerres.aacrjournals.org/content/suppl/2011/10/17/0008-5472.CAN-11-1212.DC1

http://cancerres.aacrjournals.org/content/71/23/7155.full#ref-list-1

http://cancerres.aacrjournals.org/content/71/23/7155.full#related-urls

http://cancerres.aacrjournals.org/cgi/alerts

mailto:[email protected]

http://cancerres.aacrjournals.org/content/71/23/7155


Documents

BlockadeofTGF-bSignalingbytheTGFbR-IKinaseInhibitor ... · investigated here the combination effects of radiation and LY2109761 in established human glioblastoma cell lines and in