LY2109761 | PPAR Signal

Blockade of TGF-b Signaling by the TGFbR-I Kinase Inhibitor LY2109761 Enhances Radiation Response and Prolongs Survival in Glioblastoma
Mengxian Zhang1,2,4, Susanne Kleber5, Manuel Ro€hrich2,4, Carmen Timke2,4, Na Han1, Jochen Tuettenberg5, Ana Martin-Villalba3, Juergen Debus4, Peter Peschke2, Ute Wirkner2, Michael Lahn6, and Peter E. Huber2,4

Abstract

Glioblastoma 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 a preclinical study of the antitumor effects of the TGF-b receptor (TGFbR) I kinase inhibitor LY2109761 in combination with radiotherapy. LY2109761 reduced clonogenicity and increased radiosensitivity in GBM cell
lines and cancer stem–like cells, augmenting the tumor growth delay produced by fractionated radiotherapy in a supra-additive manner in vivo. In an orthotopic intracranial model, LY2109761 signiﬁcantly reduced tumor growth, prolonged survival, and extended the prolongation of survival induced by radiation treatment. Histologic analyses showed that LY2109761 inhibited tumor invasion promoted by radiation, reduced tumor microvessel density, and attenuated mesenchymal transition. Microarray-based gene expression analysis revealed signaling
effects of the combinatorial treatments that supported an interpretation of their basis. Together, these results show that a selective inhibitor of the TGFbR-I kinase can potentiate radiation responses in glioblastoma by coordinately 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 TGFbR
kinase inhibitors as radiosensitizers to improve the treatment of glioblastoma. Cancer Res; 71(23); 7155–67. ©2011
AACR.

Introduction

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

Authors' Afﬁliations: 1Department of Oncology, Tongji Hospital, Tongji Medical College, Huazhong University of Science & Technology, Wuhan, China; Departments of 2Radiation Oncology and 3Neurobiology of Brain Tumors, German Cancer Research Center (DKFZ); 4Department of Radiation Oncology, University Hospital Center, Heidelberg; 5SHG-Klinik Idar-Oberstein Klinik fu€r Neurochirurgie, 2 Dr.-Ottmar-Kohler-Straße, Idar- Oberstein, Germany; and 6Oncology Early Clinical Investigation, Lilly Research Laboratories, Indianapolis, Indiana
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).
Corresponding Author: Peter E. Huber, Department of Radiation Oncol- ogy, German Cancer Research Center (DKFZ)/University of Heidelberg Medical 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.

cancer stem cells (CSC); or tumor-initiating cells (TIC)]. GBMs are among the ﬁrst solid cancers in which CSLCs were identiﬁed (1, 2). CSLCs markers such as CD133 and side population have been used to prospectively isolate a small fraction of cells in human brain tumors with increased potential to generate tumor neurospheres and xenografts (3, 4). Recent reports suggest that the expression of the CD133 antigen in gliomas and other brain tumors could serve as a prognostic indicator for tumor recurrence, malig- nant progression, treatment resistance, and patient survival (5). Several studies indicate that conventional radiation
therapies appear to predominantly target the better differ- entiated CD133— population while leaving many CD133þ CSLCs alive (6). Thus, targeting CSLCs using inhibitors of
TGF-b could be a promising attempt to improve glioblas-
toma treatment (7).
TGF-b is a family of polypeptides that regulates a wide variety of biologic functions including cell proliferation,
migration, survival, angiogenesis, immunosurveillance, and embryonic stem cell maintenance and differentiation. The
multifunctional 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 factors SMAD2 and SMAD3, which then binds to SMAD4 and
translocates to the nucleus, where they regulate transcrip- tion of various target genes (8). The overexpression of TGF-b

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ligands has been reported in various malignant entities including malignant gliomas (9–11). In patients with malig- nant glioma, elevated levels of TGF-b have been reported to
be associated with high tumor grade, advanced tumor
stages, and poor patient outcome (9, 12). In addition to be
implicated in invasion and intratumoral angiogenesis in glioblastoma, TGF-b signaling has also been reported to play a pivotal role in maintenance of stemness of glioma CSLCs (13, 14). These multiple roles of TGF-b in glioma initiation and progression have promoted the development of therapeutic agents based on the inhibition of the TGF-b
pathway (13, 15). LY2109761, a novel TGFbR-I kinase inhib-
itor, has shown a SMAD2-selective inhibitory proﬁle with
antitumor activity in various tumor models such as colo- rectal cancer (16), pancreatic cancer (17), and hepatocellular carcinoma (18).
Because radiotherapy is a mainstay treatment modality in glioblastoma but may promote radioresistance in glioma
stem cells (6), a combination with an inhibitor of TGF-b,
which targets in particular CSLCs (7), seems promising. We
investigated here the combination effects of radiation and LY2109761 in established human glioblastoma cell lines and in GBM CSLCs in vitro and in vivo in a subcutaneous and an orthotopic tumor model. Our data indicate that LY2109761 is an effective treatment approach alone and augments the radiation treatment response in particular in GBM CSLCs.

Materials and Methods

Cell cultures and treatment conditions
The 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 with epidermal growth factor and ﬁbroblastic growth factor, sodium pyruvate, glutamine, B27, nonessential amino acids, and penicillin/streptomycin (Gibco). NMA-23 cells were
isolated from human glioblastoma surgical sample using ﬂuorescence-activated cell-sorting (FACS) method with a phycoerythrin (PE)-labeled CD133 antibody (Miltenyi Bio- tec) as previously described (19). The collection of human biopsy tissue was approved by the regional ethical commit- tee. LY2109761 was kindly provided by Eli Lilly, constituted in dimethyl sulfoxide (10 mmol/L), and stored at 20◦C. Cell exposures with LY2109761 were conducted 2 hours prior to irradiation with 6 MV X-rays (Mevatron, Siemens) at a dose
rate of 2.5 Gy/min.

Short interfering RNA treatment
Validated Stealth RNAi (Invitrogen) speciﬁc to TGFbR-I was transfected into U87MG and NMA-23 by using Lipofectamine
RNAiMAX (Invitrogen) according to the manufacturer’s pro- tocol. Stealth RNAi negative control with low GC content (Invitrogen) was used as negative control. The expression of
TGbFR1 was measured by quantitative real-time PCR (qRT-
PCR), and phosphorylation of SMAD2 was examined using
Western blot analysis.

Clonogenic assay
Increasing 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] were
plated in 25 cm2 ﬂasks, irradiated, and returned to the
incubator for 10 to 14 days. Colonies formed were stained with crystal violet (Sigma) and those with at least 50 cells were counted by microscopic inspection. The linear qua- dratic equation was ﬁtted to data sets to generate survival curves, and dose enhancement factor for drugs was calcu- lated at 10% surviving fraction (DEF0.1).

Neurosphere formation, limiting dilution, and proliferation assay
For the neurosphere formation assay, 1,000 NMA-23 cells (pretreated with LY2109761 for 2 hours or transfected with
siRNA targeting TGbFR-I or nonsense siRNA for 48 hours) were
seeded on 25 cm2 ﬂasks and irradiated. After 7 days of
incubation, numbers of neurospheres consisting of at least 50 cells were counted under a microscope. For the limiting dilution assay, increasing numbers of NMA-23 cells were plated
in 96-well plates in 200 mL serum-free medium and treated with
radiation or LY2109761 or their combination. After 7 days of
culture, the percentage of wells not containing neurospheres for each cell-plating density was calculated and plotted against the number of cells plated per well. For the proliferation assay, 50,000 NMA-23 cells were seeded on 25 cm2 ﬂasks overnight and then treated as described, incubated for another 72 hours, when the numbers of living cells were counted.

Immunoﬂuorescent staining for gH2AX
Cells were grown and treated in chamber slides. At speciﬁed time points, cells were ﬁxed with 3% paraformaldehyde for 10 minutes and then permeabilized with 0.5% Triton X-100 for 30 minutes on ice. Antibody [AlexaFluor 488 anti-H2AX phos- phorylated (Ser139); Biolegend] was added at a dilution of 1:100 in 3% bovine serum albumin and incubated overnight at 4◦C. Cells were washed and nuclei were counterstained with 1
mg/mL 40,6-diamidino-2-phenylindole. Foci were counted by
the automated image analysis system Metacyte with a Zeiss
Axioplan-2 imaging epiﬂuorescence microscope equipped with ISIS software (MetaSystems). For each treatment condi-
tion, gH2AX foci were analyzed in at least 400 cells and median
number of foci was determined.

Apoptosis and ﬂow cytometry
Twenty-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 in PBS, 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 cells with sub-G1 DNA content. To measure caspase-3 activity, cells
were treated according to the manufacturer’s suggestions (BD Pharmingen), 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.).

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Western blot analysis
Six hours after treatment as indicated, cells were washed twice with PBS, and lysed using Qproteome Mammalian Protein 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 with antibodies against phospho (p)-SMAD2 and total SMAD2 (Cell
Signaling) with an anti-b-actin monoclonal antibody (Sigma)
as internal loading control.

Microarray analysis
U87MG cells were solubilized and homogenized in TRIzol (Invitrogen) 6 hours after radiation with or without LY2109761
treatment (4 Gy, 10 mmol/L). For LY2109761 treatment, cells
were exposed to LY2109761 for 2 hours prior to irradiation as in
the other in vitro assays. Total RNA was isolated according to the manufacturer’s instruction, purity and integrity of the RNA were assessed with Agilent 2100 BioAnalyzer (Agilent Tech- nologies), and 200 ng of total RNA was used for ampliﬁcation and 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 spin columns from QIAGEN. Quantity and cyanine incorporation of labeled cRNAs were determined using a Nanodrop ND-1000 spectrophotometer (Peqlab Biotechnologies GmbH). The labeled probes were then hybridized to an Agilent 4 44 K whole human genome microarray (G4112F) containing 45,015 features 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 statistically analyzed with SUMO software as previously described (20). Ingenuity Pathway Analysis was used for functional analysis (IPA; Ingenuity Systems, Inc.; ref. 21). Microarray data were deposited in “ArrayExpress” (Accession No.: E-TABM-1148).

qRT-PCR
To 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) values with those of housekeeping gene GAPDH.

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

twice daily (days 1–5 of each week) 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 orthotopic CSLC model in SCID mice
Beige severe-combined immunodeﬁcient (SCID) mice (8-
week-old, 20 g; Charles River Laboratories) were anesthetized and stereotactically inoculated with NMA-23 cells (104 cells in
2 mL PBS) into the left forebrain (2 mm lateral, 1 mm anterior to
bregma, at 3 mm depth from skull surface). Animals were
randomized into 4 treatment groups (control, LY2109761, radiation, radiation plus LY2109761; n 13 each, with 4 mice scheduled for histology). LY2109761 treatment (50 mg/kg twice daily) started on day 1 after tumor inoculation and was admin- istered 5 days weekly until the end of observation. Radiation was delivered on day 4 to the entire head of anesthetized mice (7 Gy single dose) using the 6 MV LINAC. On day 15, animals were examined by MRI at 1.5 T (Siemens Magnetom Vision) using a custom-made small animal solenoid Tx/Rx radiofre- quency coil. Tumor volumes were estimated using gadolinium- enhanced T1-weighted spin-echo images. A 3-dimensional reconstruction of the tumor surface and calculation of the tumor volume were conducted with the QuickVol software as previously described (22). For survival studies, moribund mice or mice with severe neurologic symptoms were euthanized.

Tumor histology and immunohistochemistry
Four animals per group were sacriﬁced on day 16 and exsanguinated by transcardial perfusion ﬁrst with ice-cold PBS. Brains were dissected, partially cryoﬁxated, or embed- ded in parafﬁn after ﬁxation in 4% paraformaldehyde for 24 hours. Sections of parafﬁn-embedded blocks were stained with hematoxylin and eosin. Immunohistochemistry was carried out with frozen sections as previously described (23). Primary antibodies included the following: anti-vimen- tin (rabbit polyclonal; Abcam; 1:200), anti-CD31 (rabbit polyclonal; 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 (rabbit polyclonal; Quidel; 1:200). After incubation with primary antibodies, the appropriate ﬂuorescence-labeled secondary antibodies were applied. Images were captured using a Nikon Eclipse E600 microscope equipped with a Nikon digital sight DS-U1 camera and subsequently analyzed using ImageJ software (NIH, Bethesda, MD). For each treatment condition, the analysis was conducted in at least 5 randomly chosen ﬁelds from 3 to 5 sections.

Statistical analysis
The unpaired 2-tailed t test was used for the comparison of parameters between groups. The Kaplan–Meier method was used to determine the median survival time (MST), and a log- rank test was used to compare the differences between survival
curves. A value of P < 0.05 was considered signiﬁcant. The
statistical analysis was conducted using the software package
Statistika 6.0 (Statsoft).

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Results

LY2109761 enhanced radiosensitivity of glioblastoma cells
To determine the effects of LY2109761 on GBM tumor cell radiosensitivity, clonogenic survival analysis was conducted. LY2109761 pretreatment reduced clonogenic survival in cell cultures of U87MG (Fig. 1A) and T98 (Fig. 1B) following radiation, resulting in an increase in the radiosensitivity with
a DEF0.1 of 1.30 and 1.37, respectively. Likewise, TGbFR-I
siRNA reduced TGbFR-I mRNA expression and inhibited
SMAD2 phosphorylation (Supplementary Fig. S1), associated
with an increase in the radiosensitivity with a DEF0.1 of 1.34 in U87MG cells (Fig. 1C), supporting the notion that
LY2109761 exhibits its radiosensitizing effect via TGF-b
signaling blockage.

LY2109761 reduced GBM-derived CSLC self-renewal and proliferation and sensitizes them to radiation
Considering that glioblastoma stem cells have been linked to radioresistance, we explored whether LY2109761 would inhibit the self-renewal of GBM CSLCs and increase the sensitivity of these TICs toward radiation. We used neurosphere formation as a surrogate marker of the clono- genic survival of glioblastoma CSLCs given they grow in
suspension (24). We found that LY2109761 (10 mmol/L) or
radiation (4 Gy) alone reduced neurosphere-forming efﬁ-
ciency in NMA-23 cells. The combination of LY2109761 plus radiation 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 siRNA alone or combined with radiation also reduced clonogeni-
city of NMA-23 cells (Fig. 2C). Furthermore, a proliferation assay revealed a reduction of NMA-23 cells proliferation/ viability after LY2109761 or radiation treatment alone, and their combination resulted in a further reduction of the cell count (Fig. 2E).

Figure 1. The effects of LY2109761 or TGFbR1 siRNA on radiosensitivity of glioblastoma cell lines measured by clonogenic survival assay. U87MG and T98 cells were pretreated with 10 and 5 mmol/L of LY2109761, respectively (which resulted in a surviving fraction of about 80%, data not
shown), 2 hours before irradiation or transfected with siRNA targeting TGbFR-I or negative nonsense siRNA for 48 hours before radiation. Colony-forming efﬁciency was determined 10 to 14 days later, and
survival curves were generated after correction for the cytotoxicity induced by LY2109761 alone and linear quadratic equation was ﬁtted to

LY2109761 enhanced radiation-induced DNA damage and apoptosis rates in glioblastoma cells and GBM- derived CSLCs
DNA damage and repair are important components of radiation-induced cytotoxicity. As a marker of recognized DNA double-strand breaks (DSB), we evaluated the induc- tion of nuclear foci of phosphorylated histone H2AX
(gH2AX). U87MG and NMA-23 cells were pretreated with LY2109761 (10 mmol/L) for 2 hours and irradiated (2 Gy). In
both cells, LY2109761 signiﬁcantly increased the number of radiation-induced gH2AX foci at 30 minutes and 24 hours postirradiation (Fig. 3A–C), whereas LY2109761 monother- apy did not signiﬁcantly alter (P > 0.5) the number of

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

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Figure 2. LY2109761 or TGFbR1 siRNA reduces NMA-23 self-
renewal and proliferation and sensitizes them to radiation. A, representative images ( 40) of neurospheres formed by NMA-23 cells treated with LY2109761 (LY;
10 mmol/L) or radiation (RT; 4 Gy) or their combination. B, the number of
neurospheres per ﬂask, compared between the control group and cells treated as indicated. C, the number of neurospheres per ﬂask, compared between the untreated group and cells treated with negative control, siRNA, RT, or siRNA RT. D, NMA-23 cell self- renewal capacity was evaluated in a limiting dilution assay under each treatment condition. The data are presented as the percentage of wells not containing neurospheres for each cell-plating density. E, NMA-23 cell proliferation was determined by cell count after 72 hours of exposure to each treatment. Relative numbers of cells
are shown as histogram. Columns, mean; bars, SD; ω, P < 0.05 versus control (or negative control);
ωω, P < 0.05 versus control
(or negative control) and each monotherapy; #, P > 0.05 versus untreated.

gH2AX foci. These data suggested that LY2109761 increased 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-hour pretreatment with LY2109761 before irradiation increased apoptosis 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 both glioblastoma cells and GBM-derived CSLCs
Because SMAD proteins are central mediators of signals from TGF-b receptors, we evaluated the effect of LY2109761 on the phosphorylation of SMAD2 (p-SMAD2), one of their imme- diate downstream targets. As expected, LY2109761 (10 mmol/L
for U87MG and NMA-23; 5 mmol/L for T98) alone or combi-
nation with radiation (4 Gy) effectively suppressed SMAD2
phosphorylation (Fig. 4).

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Figure 3. LY2109761 increases radiation-induced gH2AX foci and increases radiation-induced
apoptosis in U87MG and NMA-23 cells. A, representative images of g H2AX foci immunoﬂuorescent
staining obtained from control and
treated cells at 24 hours after radiation in U87MG. B, representative results of the foci number distribution using an automated image analysis system for treated and control cells at 24 hours after radiation in U87MG. The histogram depicts the frequency of the cells with a speciﬁc number of foci and the line scatter plot shows the accumulated percentage of cell frequency. C, the median number of foci compared between controls and treated groups in U87MG (left) and NMA-23 (right). Columns,
mean; bars, SD; ω, P < 0.05.

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

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

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Figure 3. (Continued ) D, combined treatment with LY2109761 and irradiation substantially enhanced apoptosis rates in a supra-additive manner. The percentage of apoptotic cells was determined by ﬂow cytometric analysis of cells with sub-G1 DNA content and active caspase-3 at 24 hours after treatments in U87MG (left) and NMA-23 (right). U87MG cells and NMA-23 cells were pretreated with LY2109761
(10 mmol/L) for 2 hours and irradiated (8 Gy). Columns, mean; bars, SD; ω, P < 0.05 versus control; ωω, P < 0.05 versus control and
respective single treatment. E,
representative results of ﬂow cytometric analysis for active caspase-3 using FlowJo software at 24 hours after treatments.

Further analysis using IPA of differentially regulated genes with at least 2-fold change (LY, 111 genes; RT, 36 genes; LY RT, 157 genes) revealed 8 functional categories with highly affected and enriched transcripts (Fig. 5C). The associated molecular and cellular functions are related to cellular move- ment, cellular growth and proliferation, gene expression, cell death, cell cycle, cell-to-cell signaling and interaction, cell signaling, and molecular transport. For example, the top function for LY2109761 and radiation alone or their combina- tion was associated with cellular movement, cell death, and cellular growth and proliferation.
Venn diagrams revealed the treatment-speciﬁc upregulated and downregulated genes (Fig. 5D) and also the commonly affected genes (Fig. 5D). A signiﬁcant number of genes were commonly affected under the treatment of LY2109761 and radiation plus LY2109761, whereas considerably fewer genes were commonly affected by radiation and radiation plus LY2109761, suggesting a comprehensive impact of LY2109761 on radiation response at the gene expression level when combined with radiotherapy.

LY2109761 enhanced radiation-induced tumor growth delay in a U87MG subcutaneous xenograft tumor model in BALB/c nude mice
A tumor growth delay experiment was carried out using U87MG tumors growing subcutaneously in the hind leg of nude mice. As shown in Fig. 6A, the time for 5-fold increase in tumor size (from 150 mm3 to 750 mm3) was calculated using the tumor volumes from the individual mice in each group. The mean time for 5-fold increase in tumor size increased from 11.4 days for sham-treated mice to 17.2 days for LY2109761-treated mice and 23.9 days for irradiated mice. Radiation plus

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

LY2109761 increased survival in an orthotopical CSLC glioblastoma model and enhanced antitumor activity of radiation
The effect of LY2109761 with or without radiation on intracranial CSLC (NMA-23) tumor growth was monitored by noninvasive MRI. By day 15, both LY2109761 and radiation

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

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Figure 5. The effects of each treatment on gene expression proﬁles generated from U87MG cells. A, a heatmap representing 988 transcripts was generated using 4-class t test (P 1.0E-004). Sets of differentially regulated genes from 3 biologic replicates were identiﬁed for each treatment condition (LY, P 1.0E-004; RT, P
2.0E-003; LY RT, P 1.0E-003)
compared with the gene expression proﬁle from untreated control using 2-class t test. Color intensity is assigned to ratios of gene expression; shades of red, genes that are upregulated; shades of green, genes that are downregulated; black, genes that are unchanged. B, fold changes in expression levels of genes regulated by LY2109761 (top) and those upregulated by radiation and reversed by radiation plus LY2109761 (bottom) as determined by microarray and qRT-PCR methods. C, bar graph representing functions signiﬁcantly different between treatments from IPA derived from genes with more than 2-fold change. X-axis indicated the signiﬁcant score in each functional category. Given the degree of enrichment in the functions, which is reﬂected by the signiﬁcance score (negative logarithm of the P value), the results indicate that the genes affected by treatments do not simply comprise a random list and may therefore be of potential functional relevance.

monotherapy markedly inhibited tumor growth by 43.4% and 54.3% versus controls (Fig. 6B and C), which was further reduced by the combination to 76.3%. The Kaplan–Meier survival curves showed that LY2109761 alone increased mod-

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

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Figure 5. (Continued ) D, Venn diagrams depicting the number of genes that were commonly upregulated or downregulated by different treatments. DMSO, dimethyl sulfoxide.

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 between
LY2109761 and radiotherapy in the orthotopic glioblastoma model.

LY2109761 inhibited tumor invasion and reduced tumor microvessel density
Previous studies have shown that glioblastoma cells can express the intermediate neuroﬁlament vimentin, including those tumor cells that inﬁltrate into normal parenchyma. To evaluate tumor invasiveness in intracranial tumor sections, hematoxylin–eosin and immunohistochemical staining for vimentin was done. As shown in Fig. 7A and B, sham-treated intracranial xenografts displayed peripheral invasion of the surrounding brain as single cells and cell clusters, frequently surrounded by numerous small satellite tumors. Interest- ingly, a more invasive growth pattern was observed after radiotherapy alone than controls. In contrast, tumors from mice receiving LY2109761 monotherapy or LY2109761 plus radiotherapy displayed a signiﬁcantly reduced invasion growth pattern compared with the control and radiotherapy group.
Next, we investigated tumor angiogenesis as another important process of tumor growth particularly pertinent in glioblastoma using CD31 endothelial staining and micro- vessel density (MVD) analysis. We found that LY2109761 alone (MVD: 39.2 8.4) and radiotherapy alone (47.7 10.5)

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 volumes were calculated using the formula: v length width width 0.5. Data, means SE; n 10 animals in each group. Statistical signiﬁcance is designated as:
ω, P < 0.05 versus control; ωω, P < 0.05 versus control and each monotherapy. B, representative gadolinium-enhanced T1-weighed images of orthotopic xenograft from sham-treated control animals and animals that had received LY2109761 (LY) or radiation (RT) or their combination examined by MRI 15 days after tumor injection. C, tumor volumes estimated from MRI. @, P < 0.05 versus control; @@, P < 0.05 versus control and each monotherapy. D, Kaplan–Meier survival analysis of beige SCID mice transplanted intracranially with NMA-23 cells treated with LY2109761 or radiation or their combination. Differences between survival curves were compared using a log-rank test. ω, P < 0.05 for MST.

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Zhang et al.

Figure 7. Histologic studies of glioblastoma orthotopic xenografts from mice treated with LY2109761 (LY), or radiation (RT), or both. Original magniﬁcation, 200. A, representative examples of the hematoxylin–eosin staining. B, representative examples of the immunoﬂuorescence staining of human vimentin, CD31, ﬁbronectin, COL5A1, and YKL-40. T, tumor; B, normal brain. C, quantitative analysis of CD31 staining in tumors given as MVD. For quantiﬁcation of MVD, vessels on each section were counted in 5 high-power
ﬁelds (magniﬁcation, 200). Columns, mean; bars, SD. ω, P < 0.05 versus control; ωω, P < 0.05 versus control and respective single treatment.

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

LY2109761 inhibited mesenchymal change in the CSLC orthotopic glioblastoma model
A mesenchymal character has been associated with increased malignancy, treatment resistance, and poor prog- nosis in glioblastoma. We found here that LY2109761 inhibited the mesenchymal character in our orthotopic CSLCs model. Immunohistochemical staining showed that LY2109761 alone and in combination with radiotherapy reduced the expression of 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 processes that are important for glioblastoma progression suggests that
this pathway may be a promising target for therapy. In the present study, we provide evidence that TGF-b signaling blockade 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-induced cytotoxicity and cooperated with radiation to suppress clo- nogenic survival of established glioblastoma cell lines. Mech- anistic studies revealed that LY2109761 may act in concert with radiation to enhance radiation-induced DNA damage and apoptosis. Second, LY2109761 also increased the sensi- tivity of primary GBM CSLCs derived from surgical specimens toward radiation. Third, LY2109761 enhanced radiation- induced tumor growth delay in 2 independent models: in U87MG tumors growing subcutaneously in BALB/c nu/nu mice and in orthotopic CSLC tumors growing in brains of SCID mice. In this orthotopic brain tumor model, LY2109761 increased animal survival alone and increased the radiation- induced prolongation of the animal life span.
The interesting ﬁnding that TGF-b inhibition impedes the
cellular DNA damage stress response and results in increased
radiosensitivity had been shown in mammary epithelial cells (26). While these authors reported a reduced gH2AX foci number following TGF-b inhibition, we found here an increase of radiation-induced g H2AX foci number that may be due to the difference in cell types or TGF-b inhibitors used.
Targeting CSLCs in human tumors including glioblastoma is an evolving concept for developing new treatment options in cancer. Stem cells have been reported to be responsible for the initiation, propagation, recurrence, and radioresistance of glio- mas (6). Notably, the most effective targeted therapies may have activity against CSLCs. Encouragingly, we found that LY2109761 reduced the self-renewal and proliferation capability of GBM CSLCs and also enhanced the radiosensitivity of GBM CSLCs. More importantly, LY2109761 further increased the antitumor effects of radiation and increased animal survival when GBM CSLCs were injected orthotopically into mice brains.
Invasion is another important feature of human glioblasto- ma responsible for their dismal outcomes. Although ionizing radiation is the mainstay of nonsurgical treatment in GBM, radiation may also promote migration and invasiveness of glioma cells (27), which has been attributed, in part, to

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LY2109761 and Radiation in an Orthotopic Glioblastoma Model

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 LY2109761 inhibited both constitutive and radiation-provoked tumor cell
invasion 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 LY2109761
alone and in combination with radiation reduced tumor MVD, indicating that LY2109761 has direct antimigratory and anti- angiogenic properties, which can be beneﬁcial in the context of an attenuation of unwanted side effects of radiotherapy such as certain promigratory and proangiogenic effects.
The fundamental role for mesenchymal change in promot- ing invasion, treatment response, and even CSC function in human carcinoma and GBM invasion is increasingly recog-
nized (6, 31). Recent studies have established that TGF-b is a
master regulator of epithelial-to-mesenchymal transition
(EMT) in carcinoma (32). To our best knowledge, its relevance to mesenchymal change in GBM models has not been reported.
Here, we found that the blockade of TGF-b signaling using
LY2109761 markedly reduced the expression of mesenchymal
markers in the orthotopic glioblastoma model. It is conceivable that this inhibitory effect on mesenchymal change of LY2109761, at least partly, contributes to its antimigratory capacity.
Considering that TGF-b is involved in multiple signaling
pathways, genome-wide microarray analysis was conducted to investigate potential mechanisms of LY2109761, TGF-b signal- ing, and radiation interactions. The regulated genes represent
functional classes involved in diverse biologic processes including cellular movement, cellular growth and proliferation, cell death, cell cycle, cell-to-cell signaling and interaction, and cell 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 promote invasion, to mediate tumor angiogenesis by production of VEGF, to be involved in the resistance of cancer cells against cytotoxic drugs (33), and has also been implicated in CSC function (34) as well as EMT process (35). Our data are here in line with a very recent report focusing on the effects of LY2109761 on ID1 (36). Angiopoietins (ANGPT) are ligands of the endothelial cell receptor TIE2 and have crucial roles in the tumor angiogenic switch, inﬂammation, metastasis, and lym- phangiogenesis. Increased expression of ANGPT2 and higher ANGPT2/ANGPT1 ratios in tumors correlate with poor prog- nosis in many cancers. Agents speciﬁcally targeting ANGPT1 and ANGPT2 are currently in phase II clinical trials (37). High expression levels of IAPs that inhibit apoptosis have been

associated with poor treatment response and prognosis (38). In addition, we also identiﬁed and conﬁrmed several genes upregulated by LY2109761 treatment, including RHOB, LRIG1, and ACTG2, which act as tumor suppressors and have putative roles 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 might be activated that contribute to tumor invasiveness, angiogen- esis, and radioresistance (42, 43). The set of such genes that were upregulated by radiation and reversed by LY2109761 further included AKT, ATF5, and LMO2. AKT is a major downstream effector of the phosphoinositide 3-kinase (PI3K) pathway, a potent prosurvival pathway, associated with radio- resistance of cancer cells with AKT inhibition having been shown to increase radiation effects on tumors (44). Activating transcription factor 5 (ATF5) is highly expressed in malignant glioma and plays an important role in promoting cell survival by stimulating transcription of MCL1, an antiapoptotic BCL2 family member (45). ATF5 has been shown to be a potent repressor of p53, and elevated expression of ATF5 in tumor is related to radioresistance and greater cell motility (46). The LIM-domain protein LMO2 is a speciﬁc regulator of tumor angiogenesis, which has been suggested an attractive drug target in cancer (47). Together these array data may provide additional molecular signaling hints for the beneﬁcial effects
observed after combined TGF-b signaling inhibition with
LY2109761 and radiation in the treatment of glioblastoma.
In summary, we have provided evidence that LY2109761, a speciﬁc TGFbR-I kinase inhibitor, is active against glioblasto- ma alone and enhances the antitumor efﬁcacy of radiation
both in vitro and in vivo, in particular in GBM CSLCs. Our ﬁndings rationalize further translational studies of LY2109761 or agents with similar properties alone and in combination with 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 Priority Research Program the Tumor-Vessel Interface (SPP1190), National Aeronautics and Space Administration Specialized Center of Research NNJ04HJ12G, Kom- petenzverbund Strahlenforschung (KVSF, 03NUK004A,C) of Bundesministerien fuer Bildung, Forschung und Umwelt (BMBF/BMU).
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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

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Blockade of TGF- Signaling by the TGFR-I Kinase Inhibitor
LY2109761 Enhances Radiation Response and Prolongs Survival in Glioblastoma
Mengxian Zhang, Susanne Kleber, Manuel Röhrich, et al.
Cancer Res 2011;71:7155-7167. Published OnlineFirst October 17, 2011.

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