Imatinib Radiosensitizes Bladder Cancer by Targeting Homologous Recombination

Published OnlineFirst January 9, 2013; DOI: 10.1158/0008-5472.CAN-12-1170

Cancer Therapeutics, Targets, and Chemical Biology Research

Boling Qiao1, Martin Kerr3, Blaz Groselj3, Mark T.W. Teo2, Margaret A. Knowles1, Robert G. Bristow5, Roger M. Phillips4, and Anne E. Kiltie3

Abstract Radiotherapy is a major treatment modality used to treat muscle-invasive bladder cancer, with patient outcomes similar to surgery. However, radioresistance is a signiﬁcant factor in treatment failure. Cell-free extracts of muscle-invasive bladder tumors are defective in nonhomologous end-joining (NHEJ), and this phenotype may be used clinically by combining radiotherapy with a radiosensitizing drug that targets homologous recombination, thereby sparing normal tissues with intact NHEJ. The response of the homologous recombination protein RAD51 to radiation is inhibited by the small-molecule tyrosine kinase inhibitor imatinib. Stable RT112 bladder cancer Ku knockdown (Ku80KD) cells were generated using short hairpin RNA technology to mimic the invasive tumor phenotype and also RAD51 knockdown (RAD51KD) cells to show imatinib's pathway selectivity. Ku80KD, RAD51KD, nonsilencing vector control, and parental RT112 cells were treated with radiation in combination with either imatinib or lapatinib, which inhibits NHEJ and

cell survival assessed by clonogenic assay. Drug doses were chosen at approximately IC40 and IC10 (nontoxic) levels. Imatinib radiosensitized Ku80KD cells to a greater extent than RAD51KD or RT112 cells. In contrast, lapatinib radiosensitized RAD51KD and RT112 cells but not Ku80KD cells. Taken together, our ﬁndings suggest a new application for imatinib in concurrent use with radiotherapy to treat muscle-invasive bladder cancer. Cancer Res; 73(5); 1–10. 2012 AACR.

Introduction outcome of radiation treatment in muscle-invasive bladder – Bladder cancer is the fourth most common cancer in men cancer (3 5). However, elderly patients are not always able in the United Kingdom (1). In a population-based study, to tolerate conventional chemotherapy agents when used as radiotherapy was found to be as effective as cystectomy in radiosensitizers. the treatment of muscle-invasive disease and is being For tumors with genomic aberrations/alterations, therapies increasingly required as the population ages (2). Radiother- targeted toward the expressed proteins, such as the tyrosine apy to the bladder results in acute bladder and bowel kinase inhibitors (TKI) imatinib and lapatinib, are an attractive toxicities in most patients and, more rarely, causes long- option as they do not have the myelosuppressive or neurotoxic term toxicity in which the most severe cases may require a side effects of chemotherapy, although they do cause diarrhea, fi cystectomy for alleviation of symptoms. Conventional cyto- skin rash, and very rarely lung brosis, and most are available toxic chemotherapy agents have been used to improve the as oral preparations. However, it is important that such costly agents are targeted to those patients most likely to benefit. Imatinib selectively inhibits the tyrosine kinase activity of ABL as well as several receptor tyrosine kinases: the platelet-derived 1 2 Authors' Affiliations: Sections of Experimental Oncology and Epide- growth factor receptors (PDGFR-a and -b), the stem cell factor miology and Biostatistics, Leeds Institute of Molecular Medicine, Leeds; 3Department of Oncology, Gray Institute for Radiation Oncology and (SCF) receptor (KIT), the discoidin domain receptors (DDR1 Biology, University of Oxford, Oxford; 4Institute of Cancer Therapeutics, and DDR2), and the colony-stimulating factor receptor (CSF- 5 University of Bradford, Bradford, United Kingdom; and Ontario Cancer 1R; refs. 6, 7) and is used to treat chronic myelogenous leukemia Institute, Princess Margaret Hospital and University of Toronto, Toronto, Canada (CML) and gastrointestinal stromal tumors (GIST). There are currently no clinical trials involving imatinib in bladder cancer. Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). Lapatinib is a TKI that selectively targets the EGF receptor (EGFR) and HER2 (8) and is indicated in the treatment of B. Qiao and M. Kerr contributed equally to this work. HER2-positive breast cancer. Clinically, lapatinib is primarily Corresponding Author: Dr. Anne E. Kiltie, Department of Oncology, Gray being investigated in metastatic bladder cancer. However, Institute for Radiation Oncology and Biology, University of Oxford, Old Road Campus Research Building, OXFORD OX3 7DQ, United KIngdom. there is an ongoing pilot study of neoadjuvant lapatinib before Phone: 44-1865-617352; Fax: 44-1865-617394; E-mail: cystectomy (9). [email protected] Bladder tumors express tyrosine kinases to varying extents: doi: 10.1158/0008-5472.CAN-12-1170 50% and 45% overexpress EGFR and HER2, respectively (10), 2012 American Association for Cancer Research. whereas PDGFR was reportedly expressed in approximately

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80% in a Chinese cohort, published only in Chinese. Although Ras-MAPK and PI3K-AKT), which are triggered by binding of KIT expression has been described in both upper tract tumors ligand to the transmembrane EGFR ligand-binding domain and small cell carcinomas of the bladder (11, 12), neither KIT (27, 28). These cascades are normally activated by ionizing nor ABL expression has been studied in transitional cell radiation in EGFR-overexpressing tumors, resulting in radio- carcinomas of the bladder to our knowledge. resistance (27). Moreover, they also act as radiosensitizers by Ionizing radiation causes DNA damage, including base repressing DNA repair in irradiated cells although this occurs damage, single-strand breaks (SSB), and double-strand breaks via the NHEJ pathway, through inhibition of the phosphoinosi- (DSB). Unrepaired or misrepaired DSBs are lethal, resulting in tide 3-kinase (PI3K)–mediated stimulation of DNA PKcs and cell death both in tumors and normal tissues (13, 14). Mam- by blocking of the nuclear interaction between EGFR and DNA malian cells use 2 major pathways to repair DSBs, namely PKcs normally induced by ionizing radiation [reviewed by homologous recombination and nonhomologous end-joining Baumann and colleagues (27)]. (NHEJ; refs. 15, 16). Homologous recombination is an error-free We hypothesized that in muscle-invasive bladder cancer, it pathway, predominantly used in the G2–S phase of the cell would be better to use an agent that targets the homologous cycle, which requires the sister chromatid to act as a DNA recombination pathway rather than the NHEJ pathway. This template. A major homologous recombination protein is would result in a form of "synthetic sickness" (see ref. 29 for RAD51, which is involved in ATP-dependent DNA strand recent review), where tumor cells already deficient in NHEJ exchange. RAD51 expression is increased following ionizing would have reduced homologous recombination efficiency and radiation in tumor cells and induces the formation of RAD51 repair, and thus increased ionizing radiation–induced lethal- nuclear foci at sites of DSB (17). NHEJ is the major DSB repair ity. As imatinib is known to target homologous recombination pathway used in G0 and G1 (18) and involves the DNA-binding via RAD51 (17, 25), it may be one such agent, and the com- complex Ku70/Ku80 and the DNA-dependent protein kinase bination of imatinib and radiotherapy should result in an catalytic subunit (DNA PKcs; ref. 19). In addition to the classical increased therapeutic ratio for muscle-invasive bladder cancer. DSB repair pathways, a less efficient Ku-independent pathway We therefore sought to see whether ABL, KIT, PDGFR, HER2, has been described in Ku-deficient yeast cells, which involves or EGFR are targets in bladder cancer by determining their role microhomology-mediated end-joining (MMEJ; ref. 20). in radiotherapy response. We then moved to an experimental We have previously shown the MMEJ phenotype in vitro system to test the effectiveness of therapies that might be used using cell-free extracts from muscle-invasive bladder tumors, in combination with radiotherapy to enhance radiosensitivity. and this is associated with reduced Ku–DNA binding and loss This included targeting the ABL/RAD51 and EGFR pathways. of TP53 function (21). Pucci and colleagues also showed reduced Ku–DNA binding in 5 advanced breast and muscle- Materials and Methods invasive bladder tumors (22). This error-prone repair was not Tissue samples and imunohistochemistry detected in normal human urothelial cell extracts (23), which Ninety-one formalin-fixed paraffin-embedded bladder suggests a therapeutic window that could be targeted by novel tumor biopsy samples were obtained from patients treated therapies. Negroni and colleagues (24) inhibited Ku80 expres- with radical radiotherapy for transitional cell carcinoma of the sion by RNA interference (RNAi) using short hairpin RNA bladder at the Leeds Cancer Centre (West Yorkshire, United (shRNA) in RT112 bladder cancer cells and showed increased Kingdom) from 2002 to 2005. Details of the patients and radiosensitivity and reduced Ku–DNA binding compared with radiotherapy treatments have been described previously parental RT112 vector-transfected cells. (30). Patients gave informed consent for use of their tissues The outcome of clinical radiotherapy depends, in part, upon and local ethical approval was obtained from the Leeds (East) the extent of DNA damage and how efficiently the cells can Research Ethics Committee (project 04/Q1206/62). repair this damage. Selective targeting of DNA repair pathways Antibody conditions were optimized by staining sections could increase tumor cell kill while sparing normal tissues, thus from control tissues, namely breast tumor for ABL, HER2, increasing the therapeutic ratio. ABL upregulates RAD51 gene EGFR, and PDGFR and skin tumor for KIT. Then 4-mm sections expression and imatinib reduces RAD51 protein expression from the bladder tumor specimens were heated, dewaxed, and and RAD51-chromatin binding (25) and reduces error-free hydrated in xylene, graded alcohols, and water. Antigen retriev- homologous recombination efficiency (17). Imatinib also al was achieved by boiling in EDTA (1 mmol/L at pH 8.0) or reduces the increased RAD51 expression induced by ionizing citric acid (pH 6.0) for 2 minutes of pressure cooking before radiation and reduces the associated RAD51 nuclear focus quenching endogenous peroxidase activity with 3% H2O2 for formation in glioma cell lines (25), and in bladder, pancreatic, 30 minutes. Endogenous protein-binding activity was prostate, and lung carcinoma cell lines, imatinib increases cell blocked using avidin–biotin–blocking agent (Vector) and kill in combination with ionizing radiation, due in part to normal goat serum (Dako) before incubation with primary mitotic catastrophe (17), unlike normal fibroblasts, where cell antibody: anti-ABL (1:250; NeoMarkers), anti-c-HER2 (1:200, survival is unaffected. In xenograft studies, imatinib increases Dako), anti-EGFR (1:100; Novocastra), anti-KIT (1:40; Novo- growth delay following fractionated radiotherapy in glioblas- castra), and anti-PDGFRb (1:25; Cell Signalling) diluted in toma, epidermoid, and prostate carcinoma models with no diluent (Dako) for 1 hour. Samples were then incubated for apparent increase in toxicity (17, 26). 30 minutes with secondary antibody conjugated to horse- EGFR inhibitors, such as lapatinib, can act as radiosensiti- radish peroxidase using the Dako ChemMate Detection Kit zers by targeting the intracellular signaling cascades (including (Dako). Immunoreactivity was revealed by incubation of

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sections with 3-30-diaminobenzidine (DAB) for 10 minutes the figure legends), and the cells then trypsinized and resus- before washing, taking through graded alcohols, clearing in pended in medium containing drug as required. xylene and counterstaining with hematoxylin (VWR) before mounting in dibutylphthalate xylene (Leica). Digital images Clonogenic assays were captured within invasive tumor areas (3–10 images per Following the necessary treatments, cells were plated at slide, 400 magnification) using an Olympus BX50 micro- appropriate cell numbers in triplicate in 10-cm culture dishes scope and c-3030 camera. containing 10 mL of fresh medium, with or without drug as Assessment was made of the percentage and intensity of required (see relevant figure legends). After 14 days incubation, tumor cells with membranous staining based on the recom- the cells were stained with 1% methylene blue (Sigma) in 50% mendations for interpretation of the HercepTest: gastric ethanol, and colonies with more than 50 cells were counted. cancer (31). Briefly, for all antibodies studied, complete, baso- The surviving fraction was determined as the total number of lateral, or lateral membrane staining was scored as an intensity colonies formed divided by the total number of cells plated of 3þ for strong intensity and 2þ for weak to moderate multiplied by the plating efficiency, as determined in untreated intensity, where at least 10% of the tumors cells stained cells. Radiation survival curves were plotted after normaliza- positive. Partial membrane staining or only faint/barely per- tion for the cytotoxicity induced by control or drug alone, in ceptible intensity staining in at least 10% of tumor cells was GraphPad Prism, using the linear-quadratic model with the scored as a 1þ. Staining intensity was scored independently in equation SF ¼ exp (aD þ GbD2). Each point on the survival a blinded manner by 2 observers, discordant scores were curve represents the mean surviving fraction from at least reviewed, and a consensus was reached. 3 independent experiments. Cause-specific survival was defined from day 1 of radiotherapy until death from bladder cancer. Death from another cause RNA interference using short hairpin RNA was considered a censored observation. Kaplan–Meier curves The siRNA expression vector pSilencer 2.1-U6 neomycin were plotted for cause-specific survival and the log-rank (Ambion), which contains a human U6 RNA polymerase III statistic used to compare survival times across categories of promoter able to transcribe shRNAs, was used in these experi- protein expression. ments. Two constructs were made for each of Ku80 and RAD51 (see Supplementary Table S1 and Supplementary Fig. S1), Reagents whereby complementary oligonucleotides were used to encode Imatinib was a generous gift from Novartis Pharma AG hairpin siRNA inserts, designed to target a 21-mer sequence of (Switzerland) and was later purchased from Stratech Scientific human Ku80 coding region or 30-untranslated region (UTR) Ltd.; lapatinib was a generous gift from GlaxoSmithKline plc. mRNA, or RAD51 coding region mRNA, respectively. These were For cell culture experiments, imatinib and lapatinib were designed using the Ambion Insert Design Tool for pSilencer dissolved in dimethyl sulfoxide (DMSO; Sigma) to a stock vectors and purchased from Sigma, and were then annealed and concentration of 10 mmol/L and stored in single-use aliquots ligated into the linearized pSilencer vector. Circular negative at 20C. control pSilencer neovector, that expresses a hairpin siRNA with limited homology to any known sequence in the human genome, Cell culture conditions was used in experiments as a nonspecific negative control. The TP53 wild-type RT112 bladder transitional cell carci- The target sequences in the human Ku80 or RAD51 gene noma cell line has been authenticated in M.A. Knowles's were determined empirically and analyzed by BLAST search, to laboratory by extensive genomic analysis [microsatellite typ- confirm a lack of homology to other coding sequences, as per ing, conventional karyotypic analysis, multiplex FISH (MFISH), manufacturer's recommendations. The target sequences of array-based copy number analysis]. Cells were grown in RPMI- clones used for clonogenic assays were Ku80 50-AAC TCC ATT 1640 (Sigma) supplemented with 10% v/v FBS (Sigma) and 2 CCT GGT ATA GAA-30 (Ku80 coding region target sequence 1) 0 0 mmol/L L-glutamine (Sigma) in a humidified atmosphere and for RAD51 5 -AAT CAC TAA TCA GGT GGT AGC-3 containing 5% CO2 at 37 C. Exponentially growing cells were (RAD51 coding region target sequence 2; Supplementary Table used in all experiments. S1). The corresponding targeting oligonucleotide sequences were for Ku80: top strand 50-GAT CCG CTC CAT TCC TGG TAT Cell irradiation AGA ATT CAA GAG ATT CTA TAC CAG GAA TGG AGT TTT Cells were harvested from exponential-phase cultures and TTG GAA A-30 and bottom strand 50-AGC TTT TCC AAA AAA diluted to 1,000 cells/mL. Five milliliter cell suspensions were CTC CAT TCC TGG TAT AGA ATC TCT TGA ATT CTA TAC then irradiated in tubes at a dose-rate of 1.0 Gy/min using an X- CAG GAA TGG AGC G-30, and for RAD51: top strand 50-GAT ray machine (Irradiator 320, NDT Equipment Services Ltd.), or CCG TCA CTA ATC AGG TGG TAG CTT CAA GAG AGC TAC cesium-137 source at 1.12 Gy/min using a Gamma-Service CAC CTG ATT AGT GAT TTT TTG GAA A-30 and bottom Medical GmbH GSR D1 irradiator. The cells were then replated strand: 50-AGC TTT TCC AAA AAA TCA CTA ATC AGG TGG into 10-cm dishes at appropriate cell densities. TAG CTC TCT TGA AGC TAC CAC CTG ATT AGT GAC G-30.

Chemosensitivity studies Stable transfection Exponentially growing cells were incubated in 75 cm2 flasks RT112 cells were seeded into 75-cm2 flasks and the following for 24 hours at appropriate drug concentrations (specified in day transfected at 60% confluence with pSilencer-Ku80,

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pSilencer-RAD51, or circular negative control pSilencer neo- using the Li-cor Odyssey Infrared Detection System (Li-cor vector, using Lipofectamine 2000 (Invitrogen), according to the Biosciences UK Ltd.). Quantification was based on normali- manufacturer's instructions. After 24 hours, fresh medium was zation to b-actin. The immunoblotting experiments were added containing the selection reagent G418 (600 mg/mL; carried out at least 3 times. Gibco, Invitrogen). Selection was continued for 14 days, with the medium refreshed every other day. Single clones were Cell-cycle analysis picked and tested for Ku80 and RAD51 expression, respectively, Propidium iodide (PI) staining and flow cytometry were by Western blotting and subsequently tested for ionizing used to determine cell-cycle stages. Cells from the batches used radiation sensitivity using clonogenic assays. for clonogenic assays were washed with PBS, fixed in ice-cold 70% ethanol, and stored at 4C before analysis. Cells were spun Western blot analysis down and resuspended in PI solution (50 mg/mL PI, 0.5 mg/mL Cells were lysed on ice in radioimmunoprecipitation assay RNase; Applied Biosystem) and incubated at room tempera- (RIPA) buffer (Sigma) with 1% of protease inhibitor and ture in the dark at least for 30 minutes. DNA content was phosphatase inhibitor (Sigma). The cells were allowed to swell detected by flow cytometry (Beckman FACScan system). The on ice for 20 minutes; then the lysate was centrifuged for 30 relative proportions of cells in the G1, S, and G2–M phases of the seconds at 12,000 g. The supernatant was carefully removed cell cycle were determined using ModFit LT 3.2 software. and stored at 20C. Total protein concentration in cell lysates was determined by the method of Bradford (Sigma). Thirty to Statistical analysis 50 mg of protein was resolved on 4% to 20% polyacrylamide gels All statistical analyses were conducted using SPSS16.0 soft- and transferred onto nitrocellulose membranes. The resulting ware. Clonogenic assays were conducted in triplicate at least 3 membranes were incubated with blocking buffer (Li-cor) and times, with the results expressed as mean þ SD as appropriate. primary antibodies. The antibodies used were rabbit polyclon- Western blot analyses were conducted at least 3 times inde- al ABL (2862; Cell Signalling), rabbit polyclonal Rad51 pendently. Results were considered statistically significant at a (ab63801; Abcam), rabbit polyclonal EGFR (sc-03; Santa Cruz P < 0.05, using a two-tailed unpaired Student t test. Sensitizer Biotechnology), mouse monoclonal anti-Ku80 (Ab-2; Neomar- enhancement ratios (SER) were calculated at a surviving kers), mouse monoclonal b-tubulin (clone TUB2.1 TT4026; fraction of 0.1 (10% survival). Cause-specific survival was Sigma), and mouse monoclonal b-actin (ab8226; Abcam). defined from day 1 of radiotherapy until death of the patient Fluorochrome-conjugated secondary antibodies (Li-cor) from bladder cancer. Death from another cause was consid- were used and detected by infra red scanning densitometry ered a censored observation. Kaplan–Meier curves were

75 µm 75 µm 75 µm

Figure 1. Immunohistochemistry studies. A, representative images, at high-power magniﬁcation (40): ABL, 1þ,2þ, and 3þ; EGFR, 1þ,2þ, and 3þ; HER2, 1þ,2þ, and 3þ; KIT-negative and -positive samples; PDGFR-b example of blood vessel staining but absent tumor cell staining. Scale bars represent 75 mm lengths. B, Kaplan-Meier survival curves from radiotherapy for: (i) ABL scoring comparing 3þ (solid line) and 2þ or lower (dashed line); (ii) EGFR scoring comparing 3þ (solid line) and 2þ or lower (dashed line); and iii) HER2 scoring comparing 3þ (solid line) and 2þ or lower (dashed line).

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100 100

80 80

Figure 2. Clonogenic assays 60 60 following 14 days of treatment with varying concentrations of imatinib 40 40 (Stratech) or lapatinib. Bars, mean of Survival (%) Survival (%) at least 3 independent experiments 20 20 þ SD. 0 0 0510 15 20 0 2 4 6 8 Concentration imatinib (μmol/L) Concentration lapatinib (μmol/L)

plotted for cause-specific survival and the log-rank statistic Inhibition of Ku80 or RAD51 expression in RT112 cells by used to compare survival times across categories of protein siRNA expression. To generate NHEJ-deficient and homologous recombination–deficient RT112 cells, we used a siRNA-based strategy to Results reduce Ku80 or RAD51 expression in RT112 cells. We tested the effectiveness of 2 21-mer siRNAs targeting different sites within Expression of tyrosine kinases in bladder tumor samples 0 We sought to estimate the percentage of muscle-invasive the exons or 3 -UTR regions of Ku80 and RAD51 (Supplemen- bladder tumors expressing the tyrosine kinases of interest tary Table S1), using pSilencer2.1-U6 neomycin, which drives (imatinib: ABL, KIT, and PDGFR-b; lapatinib: HER2 and EGFR), expression of a shRNA from the human U6 promoter. The on the basis that this is likely to represent the cohort of patients hairpin RNA is then processed into an siRNA, which induces for which the addition of a TKI to their radiotherapy may be RNAi of the target gene. RT112 cells were stably transfected beneficial. We also wished to determine whether these factors with each of the vectors and clones picked and tested using were prognostic in these patients, who had not received such Western blotting and ionizing radiation clonogenic assays. An treatments in addition to radiotherapy. The Dako scoring individual clone for each of Ku80 (C13, coding region) and system for the HercepTest was applied to our tyrosine kinases RAD51 (795J) was then selected for drug-ionizing radiation of interest in 91 formalin-fixed paraffin-embedded bladder clonogenic assays. Densitometric analysis of representative tumor samples (Fig. 1A). A sample was classified as positive if there was membranous staining in at least 10% of cells, which met the threshold criteria for intensity of staining (2þ or 3þ). In 75 of 91 (82%) cases there was positive immunostaining for ABL. In contrast, KIT expression was undetectable in most of the cases but showed weak staining in 8 cases, and PDGFR-b was expressed in endothelial cells and smooth muscle cells but was undetectable in bladder tumor cell membranes. There were 87 of 91 cases (96%) positive for EGFR staining and 86 of 90 (96%) cases HER2-positive. We also correlated tyrosine kinase expression with patient survival, to look for prognostic significance of high expression. We classified patients into those with low tumor tyrosine kinase expression (equal or less than 2þ) and patients with high (3þ) expression levels (as there were insufficient tumors scoring 0/1þ for meaningful comparison with 2þ/3þ). Neither ABL, EGFR, nor HER2 were significantly correlated with patient survival (Fig. 1B).

Effects of imatinib and lapatinib on RT112 cell proliferation ﬁ Before combining drug and radiation treatments, it was rst Figure 3. Clonogenic assays following ionizing radiation (IR) in RT112 necessary to determine the growth-inhibitory activity of the parent cells (RT112), and clones selected under G418 from RT112 cells drugs in RT112 cells. Figure 2 shows cell viabilities by clono- transfected with circular negative control pSilencer neovector genic assay following 14 days treatment with varying concen- (pSilencer), RT112 cells transfected with Ku80-construct (Ku80KD), and RT112 cells transfected with RAD51-construct (RAD51KD). Bars, mean trations of imatinib or lapatinib. The IC50 value for imatinib in þ ﬁ m m of at least 3 independent experiments SD. Statistical signi cance was RT112 cells was 8 mol/L and for lapatinib 3.5 mol/L. determined by two-tailed, unpaired Student t test (P ¼ 0.01).

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Western blot analyses revealed a 30% reduction in Ku80 drug, before irradiation and plating followed by 14 days incu- protein expression in C13 cells and 75% reduction in RAD51 bation, before staining and counting. Drug doses were chosen expression in 795J cells compared with parental RT112 cells at approximately IC40 (high) and at IC10 nontoxic (low) levels. and pSilencer neovector negative control cells (Supplementary Radiation survival curves were generated for each cell line Fig. S1). after normalization for the level of cell killing induced by drug alone. As shown in Fig. 4A–C, 3 mmol/L imatinib had a Ku80 or RAD51 interference causes a decreased viability significant radiosensitizing effect on Ku80KD cells (SER 1.27; of RT112 after X-ray exposure P ¼ 0.03) but no significant effect in parental RT112 cells (SER We examined the radiosensitizing effect of siRNA-mediated ¼ 1.07; P ¼ 0.051) or RAD51KD cells. At 6 mmol/L concentra- downregulation of Ku80 and RAD51 expression in RT112 cells tion, equivalent to approximately IC40, imatinib produced an using clonogenic assays. We found a statistically significant SER of 1.69 in Ku80KD cells (P ¼ 0.03) but only an SER of 1.34 in increase in radiosensitivity for the Ku80KD (P ¼ 0.01) and the RT112 parental cell line (P ¼ 0.03; P ¼ 0.046 compared with RAD51KD (P ¼ 0.02) cells as compared with parental RT112 Ku80KD 6 mmol/L imatinib survival curve) and 1.11 in the and RT112-pSilencer vector control cells (Fig. 3). RAD51KD cells (P ¼ 0.03; P ¼ 0.04 compared with Ku80KD 6 mmol/L imatinib survival curve). Imatinib significantly radiosensitized Ku80KD RT112 In contrast, lapatinib radiosensitized RT112 cells in a dose- cells dependent manner (Fig. 4D; SER ¼ 1.13 and 1.30 for 0.6 mmol/L; We then tested our hypothesis that imatinib radiosensitizes P ¼ 0.02 and 2.4 mmol/L; P ¼ 0.01) but had no radiosensitizing NHEJ-deficient Ku80KD cells, whereas having less effect on the effect on Ku80KD cells at either low (0.6 mmol/L) or high (2.4 parental RT112 cells with an intact NHEJ pathway, due to mmol/L) concentrations (Fig. 4E). However, in contrast to targeting the ionizing radiation-induced increase in RAD51. imatinib, lapatinib had a dramatic radiosensitizing effect in We conducted clonogenic assays on RT112, Ku80KD, and RAD51KD cells, even at low dose (SER ¼ 1.27; P ¼ 0.02), with RAD51KD cells using relevant drug–ionizing radiation combi- the high dose resulting in inhibition of RAD51KD cell colony nations. Cells were incubated for 24 hours with or without formation, even without ionizing radiation (Fig. 4F).

ABC 1 1 1

0.1 0.1 0.1

0.01 0.01 0.01 RT112 Ku KD Rad51 KD Surviving fraction RT112 + 3 μmol/L imatinib Surviving fraction Ku KD + 3 μmol/L imatinib Surviving fraction Rad51 KD + 3 μmol/L imatinib RT112 + 6 μmol/L imatinib Ku KD + 6 μmol/L imatinib Rad51 KD + 6 μmol/L imatinib 0.001 0.001 0.001 0246810 0246810 0246810 Dose IR (Gy) Dose IR (Gy) Dose IR (Gy) DEF 1 1 1

0.1 0.1 0.1

0.01 0.01 0.01 RT112 Ku80 KD μ

Surviving fraction RT112 + 0.6 mol/L lapatinib Surviving fraction Surviving fraction Ku80 KD + 0.6 μmol/L lapatinib Rad51 KD RT112 + 2.4 μmol/L lapatinib Ku80 KD + 2.4 μmol/L lapatinib Rad51 KD + 0.6 μmol/L lapatinib 0.001 0.001 0.001 0246810 0246810 0246810 Dose IR (Gy) Dose IR (Gy) Dose IR (Gy)

Figure 4. Clonogenic survival of cells under ionizing radiation (IR) with or without drug exposure. The radiation survival curves were generated for each cell line after normalization to cell killing by drug alone. Bars, mean survival of at least 3 independent experiments þ SD. A, RT112, ionizing radiation/imatinib (3 mmol/L, P ¼ 0.051; 6 mmol/L, P ¼ 0.03); B, Ku80KD, ionizing radiation/imatinib (3 mmol/L, P ¼ 0.03; 6 mmol/L, P ¼ 0.03); C, RAD51KD, ionizing radiation/ imatinib (3 mmol/L, P ¼ 0.08; 6 mmol/L, P ¼ 0.03). D, RT112, ionizing radiation/lapatinib (0.6 mmol/L, P ¼ 0.02; 2.4 mmol/L, P ¼ 0.01); E, Ku80KD, ionizing radiation/lapatinib (0.6 mmol/L, P ¼ 0.60; 2.4 mmol/L, P ¼ 0.86); F, RAD51KD, ionizing radiation/lapatinib (0.6 mmol/L, P ¼ 0.02).

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Cell-cycle effects of imatinib and lapatinib the RAD51KD cells. HER2 was not detectable in any of the cell To determine whether the drug-mediated enhancement of lines. We found no major increase in RAD51 levels following 8 radiosensitivity was due to cellular synchronization into a Gy ionizing radiation. Imatinib treatment was associated with radiosensitive phase of the cell cycle, PI staining and ﬂow reduced RAD51 levels in RT112 and Ku80KD cells both alone cytometry were used to determine the cell-cycle phase distri- and following 8 Gy ionizing radiation but no effect on ABL bution of samples used in each independent clonogenic assay. levels; lapatinib had no effect on RAD51 levels. Neither imatinib nor lapatinib caused obvious cell-cycle arrest in treated cells of each cell type, except for a small increase in Discussion G1 fraction in RAD51KD cells treated by lapatinib at both drug concentrations (Fig. 5; P ¼ 0.02 low dose; P ¼ 0.04 high dose). Radiotherapy is a valid option in the radical treatment of muscle-invasive bladder cancer, with similar survival rates to Effects of imatinib and lapatinib on RAD51 and EGFR cystectomy in our recent study (2). Cytotoxic chemotherapy is We also measured expression of ABL, EGFR, HER2, and often combined with radiotherapy to improve survival rates RAD51 using Western blotting (Fig. 6). Cells were incubated (3–5), but this can be at the expense of late side effects. with or without drug for 24 hours and then irradiated to 8 Gy or Moreover, many patients with bladder cancer who elect to left untreated. Forty-eight hours later, cells were lysed for have radiotherapy are elderly with poor renal function, and Western blotting. RT112, Ku80KD (C13) and RAD51KD therefore cannot tolerate cisplatin-based chemotherapy regi- (759J) cells all had detectable baseline levels of ABL, EGFR, mens. There is therefore an urgent need to identify less toxic and RAD51 although RAD51 levels were markedly reduced in agents suitable for these patients.

RT112 DMSO 80 80 RT112 RT112 3 μmol/L IM RT112 0.6 μmol/L LP RT112 6 μmol/L IM 60 60 RT112 2.4 μmol/L LP

40 40

20 20 Percentage (%) Percentage (%) Percentage

0 0 G1 SGG2–M 1 S G2–M Cell-cycle phase Cell-cycle phase

80 Ku80 KD DMSO 80 Figure 5. Cell-cycle phase Ku80 KD 3 μmol/L IM Ku80KD distribution in cells with or without Ku80 KD 6 μmol/L IM 60 Ku80KD 0.6 μmol/L LP drug exposure for 24 hours. Column, 60 μ mean of at least 3 independent Ku80KD 2.4 mol/L LP experiments; error bars represent 40 40 SD. A, RT112, Ku80KD, and RAD51KD cells treated with or 20 20 without imatinib (Stratatech) for 24 (%) Percentage (%) Percentage hours; B, RT112, Ku80KD and RAD51KD cells treated with or 0 0 without lapatinib for 24 hours. G1 S G2–M G1 S G2–M Cell-cycle phase Cell-cycle phase

80 RAD51 KD DMSO 80 RAD51KD RAD51 KD 3 μmol/L IM RAD51KD 0.6 μmol/L LP 60 RAD51 KD 6 μmol/L IM 60 RAD51KD 2.4 μmol/L LP

40 40

20 20 Percentage (%) Percentage (%) Percentage

0 0 G1 SGG2–M 1 S G2–M Cell-cycle phase Cell-cycle phase

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A RT112 C13 RT112 795J 8 Gy +- - +++- - ++- - + - - + Imatinib --++ --++ --++ --++ c-Abl

Ku80 Figure 6. Western blot analyses of ABL, EGFR, and RAD51 after 12 m 11.6 mmol/L) Rad51 mol/L (Novartis, IC50 imatinib treatment (A) and 3.5 mmol/L lapatinib treatment (B). β -Tubulin Cells were incubated with or RT112 C13 RT112 795J without drug for 24 hours and then B irradiated to 8 Gy or left untreated. 8 Gy +- - +++- - ++- - + - - + Cells were lysed 48 hours later. No Lapatinib --++ --++ --++ --++ HER2 was detectable. Full-length EGFR blots in Supplementary Fig. S2.

Ku80

Rad51

β-Tubulin

Our immunohistochemistry data show that ABL is ABL expression levels (Fig. 6) in both parental RT112 cells and expressed in more than 80% of muscle-invasive bladder Ku80KD cells, imatinib treatment was associated with reduced tumors, suggesting that imatinib might be useful in such RAD51 expression levels both with and without ionizing radi- patients. EGFR and HER2 staining was positive in more than ation. As imatinib had no radiosensitizing effect at low dose 95% of bladder tumors. While the Kaplan–Meier survival and only limited effects at high dose in RAD51KD cells, this curves (Fig. 1B) showed no prognostic significance, our sample seems to support our hypothesis that imatinib works through numbers were relatively small, with only 73% power to detect a the homologous recombination pathway but not the NHEJ HR of 0.4, with 35 cause-specific survival events at P ¼ 0.05. pathway. In contrast, lapatinib had no radiosensitizing effect Also, we used a scoring system developed for another tumor on Ku80KD cells but a marked effect in RAD51KD cells and also type and have not validated our findings in an independent sensitized parental RT112 cells, consistent with lapatinib patient cohort, so results should be treated with caution. As we acting via NHEJ rather than homologous recombination. did not have access to a patient cohort treated with a TKI and Recently, Myllynen and colleagues (33) showed that both radiotherapy, we cannot comment on the potential predictive homologous recombination and NHEJ are involved in regula- value of such markers. tion of DSB repair by EGFR, using an I-SceI–based reporter We established that both imatinib and lapatinib are cyto- system, with reduction of homologous recombination by the toxic in the RT112 bladder cancer cell-line, which has modest TKI erlotinib at 0.5 mmol/L and less so by the monoclonal ABL and EGFR expression levels (Fig. 2). Choudhury and antibody cetuximab at 30 nmol/L concentration. However, our colleagues (17) found an IC50 value of 20 mmol/L for imatinib experiments suggest that lapatinib does not act on homolo- in RT112 cells, whereas ours was 8 mmol/L; McHugh and gous recombination at 0.6 or 2.4 mmol/L. Other than a small colleagues found the IC50 for lapatinib in RT112 cells was effect on G1 arrest for lapatinib at both concentrations in 1.1 mmol/L MTT assay (32), ours being 3.5 mmol/L on clono- RAD51KD cells, neither drug affected cell-cycle progression at genic assay, perhaps reflecting differences in experimental 24 hours. Lapatinib has been found to induce G1 arrest in – systems. We then successfully knocked-down both Ku80 and bladder cancer (RT112, 45% 65% G1 cells after 72 hours of 1.1 m RAD51, using shRNA technology, in TP53 wild-type RT112 cell mol/L lapatinib; ref. 34) and gastric cancer cell lines (58% G1 lines (Fig. 3). Negroni and colleagues (24) used the same to 70% after 24 hours of 1 mmol/L lapatinib; ref. 35). Treatment method to knockdown Ku80 in RT112 cells and showed with imatinib (5–6 mmol/L) for 48 to 72 hours caused a slight – radiosensitivity and reduced Ku DNA binding. We previously increase in the number of cells in G1-phase in head-and-neck observed the latter in muscle-invasive bladder tumor extracts. squamous carcinoma cell lines (47% G1 to 58%; ref. 36), ovarian We then showed the radiosensitizing effects of both imatinib cancer cell lines (80% G1 to 94%; ref. 37) but not in small-cell and lapatinib in RT112, Ku80KD, and RAD51KD cells (Fig. 4). lung carcinoma cell lines (38). Imatinib was an effective radiosensitizer in Ku80KD cells but Our data support the role of DNA repair in both imatinib and less effective in parental RT112 cells. Although it did not affect lapatinib's radiosensitizing effects. They suggest that imatinib

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Imatinib as Radiosensitizer in Bladder Cancer

may be a useful radiosensitizer in muscle-invasive bladder Writing, review, and/or revision of the manuscript: B. Qiao, M. Kerr, B. Groselj, M.T.W. Teo, M.A. Knowles, R.G. Bristow, R.M. Phillips, A.E. Kiltie cancer, as our tumors so far have all shown the MMEJ phe- Administrative, technical, or material support (i.e., reporting or orga- notype, with defective Ku–DNA binding and defective TP53 nizing data, constructing databases): B. Qiao, M.T.W. Teo function, whereas superficial tumors had intact NHEJ (21). We Study supervision: A.E. Kiltie are currently testing further tumor sample extracts. If some muscle-invasive tumors show intact NHEJ, imatinib would not Acknowledgments be appropriate as a radiosensitizer in these patients, and there The authors thank Ms. Filomena Estevez for her expert help with immunohistochemistry staining and Drs. Sameer Chilka for outlining the muscle-invasive would be a need to develop a preradiotherapy predictive end- tumor areas, Paul Manley of Novartis and Daniel Ridley of Glaxo Smith Kline for joining assay for use in patients with muscle-invasive bladder critical reading of the article. cancer. An advantage of imatinib as a radiosensitizer in this context, unlike lapatinib, is that we would expect normal tissue Grant Support sparing and an increase in therapeutic ratio. This study was supported by Yorkshire Cancer Research project grant L336 and Cancer Research UK; Yorkshire Cancer Research Project Grant L336 (B. fl Qiao); Slovene Human Resources Scholarship Fund (B. Groselj); Yorkshire Disclosure of Potential Con icts of Interest Cancer Research Project Grant L350 (M.T.W. Teo); Cancer Research UK Program fl No potential con icts of interest were disclosed. grant C6228/A7675 (M.A. Knowles); Canadian Foundation for Innovation and Canadian Cancer Society and a Canadian Cancer Research Society Career Authors' Contributions Scientist Award (R.G. Bristow); and Cancer Research UK Program Grant Conception and design: B. Qiao, M. Kerr, R.M. Phillips, A.E. Kiltie C5255/A12678 (M. Kerr and A.E. Kiltie). Development of methodology: B. Qiao, M. Kerr, R.G. Bristow, A.E. Kiltie The costs of publication of this article were defrayed in part by the payment of Acquisition of data (provided animals, acquired and managed patients, page charges. This article must therefore be hereby marked advertisement in provided facilities, etc.): B. Qiao, A.E. Kiltie accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): B. Qiao, M. Kerr, B. Groselj, M.T.W. Teo, R.M. Phillips, Received April 2, 2012; revised December 7, 2012; accepted December 18, 2012; A.E. Kiltie published OnlineFirst January 9, 2013.

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OF10 Cancer Res; 73(5) March 1, 2013 Cancer Research

Imatinib Radiosensitizes Bladder Cancer by Targeting Homologous Recombination

Boling Qiao, Martin Kerr, Blaz Groselj, et al.

Cancer Res Published OnlineFirst January 9, 2013.

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