Cetuximab Induces Eme1-Mediated DNA Repair

Volume 16 Number 3

March 2014

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Cetuximab Induces Eme1-Mediated DNA Repair: a Novel Mechanism for Cetuximab Resistance1,2,3,4

Agnieszka Weinandy*, ¶, Marc D. Piroth†, Anand Goswami*, Kay Nolte*, Bernd Sellhaus*, Jose Gerardo-Nava*, Michael Eble†, Stefan Weinandy‡, Christian Cornelissen§, 3, Hans Clusmann¶, Bernhard Lüscher§, 4 and Joachim Weis*, 4 *Institute of Neuropathology, Medical Faculty, RWTH Aachen University and JARA-BRAIN (Jülich Aachen Research Alliance Brain) Translational Medicine, Aachen, Germany; † Department of Radiation Oncology, Medical Faculty, RWTH Aachen University and JARA-BRAIN Translational Medicine, Aachen, Germany; ‡ Department of Tissue Engineering and Textile Implants, Applied Medical Engineering-Helmholtz Institute for Biomedical Engineering, Medical Faculty, RWTH Aachen University and JARA-BRAIN Translational Medicine, Aachen, Germany; § Institute of Biochemistry and Molecular Biology, Medical Faculty, RWTH Aachen University and JARA-BRAIN Translational Medicine, Aachen, Germany; ¶ Department of Neurosurgery, Medical Faculty, RWTH Aachen University and JARA-BRAIN Translational Medicine, Aachen, Germany

Abstract Overexpression of the epidermal growth factor receptor (EGFR) is observed in a large number of neoplasms. The monoclonal antibody cetuximab/Erbitux is frequently applied to treat EGFR-expressing tumors. However, the application of cetuximab alone or in combination with radio- and/or chemotherapy often yields only little benefit for patients. In the present study, we describe a mechanism that explains resistance of both tumor cell lines and cultured primary human glioma cells to cetuximab. Treatment of these cells with cetuximab promoted DNA synthesis in the absence of increased proliferation, suggesting that DNA repair pathways were activated. Indeed, we observed that cetuximab promoted the activation of the DNA damage response pathway and prevented the degradation of essential meiotic endonuclease 1 homolog 1 (Eme1), a heterodimeric endonuclease involved in

Abbreviations: BRCA1, breast cancer tumor suppressor 1; BrdU, bromodeoxyuridine (5-bromo-2'-deoxyuridine); Chk, checkpoint kinase; DDR, DNA damage response; EGFR, epidermal growth factor receptor; Eme1, essential meiotic endonuclease 1 homolog 1; Erk, extracellular signal-regulated kinase; GBM, glioblastoma multiforme; Mus81, methyl methanesulfonate–sensitive UV-sensitive 81; STAT3, signal transducer and activator of transcription 3 Address all correspondence to: Dr Rer nat Agnieszka Weinandy, Institute of Neuropathology and Department of Neurosurgery, Medical Faculty, RWTH Aachen University, Pauwelsstr. 30, 52074 Aachen, Germany. E-mail: [email protected]; [email protected] 1 Funding: The study was funded by the START program “Molecular tumor markers and their function,” Medical Faculty, RWTH Aachen University (Aachen, Germany). Conflict of interest statement: We declare that this manuscript is original, has not been published before, and is not currently being considered for publication elsewhere. We confirm that there are no known conflicts of interest associated with this publication and that there has been no significant financial support for this work that could have influenced its outcome. We confirm that the manuscript has been read and approved by all named authors and that there are no other persons who satisfied the criteria for authorship but are not listed. We confirm that we have given due consideration to the protection of intellectual property associated with this work and that there are no impediments to publication, including the timing of publication, with respect to intellectual property. In so doing, we confirm that we have followed the regulations of our institutions concerning intellectual property. 2 This article refers to supplementary materials, which are designated by Figures W1 to W7 and are available online at www.neoplasia.com. 3 Present address: Novartis Pharma GmbH, Roonstr. 25, 90429 Nürnberg, Germany. 4 Authors with equal contribution. Received 13 September 2013; Revised 14 February 2014; Accepted 17 February 2014 Copyright © 2014 Neoplasia Press, Inc. All rights reserved 1476-5586/14/$25.00 http://dx.doi.org/10.1016/j.neo.2014.03.004

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DNA repair. The increased levels of Eme1 were necessary for enhanced DNA repair, and the knockdown of Eme1 was sufficient to prevent efficient DNA repair in response to ultraviolet-C light or megavoltage irradiation. These treatments reduced the survival of tumor cells, an effect that was reversed by cetuximab application. Again, this protection was dependent on Eme1. Taken together, these results suggest that cetuximab initiates pathways that result in the stabilization of Eme1, thereby resulting in enhanced DNA repair. Accordingly, cetuximab enhances DNA repair, reducing the effectiveness of DNA-damaging therapies. This aspect should be considered when using cetuximab as an antitumor agent and suggests that Eme1 is a negative predictive marker. Neoplasia (2014) 16, 207–220.e4

Introduction Overexpression of the epidermal growth factor receptor (EGFR) is found in many tumors and has been linked to increased cell proliferation, angiogenesis, and decreased apoptosis [1]. Diverse strategies have been established to target and inhibit the EGFR in tumors, including monoclonal antibodies and tyrosine kinase inhibitors [2]. Cetuximab (Erbitux; Merck KgAa, Darmstadt, Germany; IMC-C225) is a recombinant, chimeric human-murine IgG1 antibody, which was developed to inhibit growth of tumor cells by binding to the extracellular domain of the EGFR, thus preventing ligand binding and subsequent activation of downstream signal transduction pathways [3]. However, the use of this monoclonal antibody alone provides response rates of only about 11% in advanced, pretreated, and recurrent malignancies [4]. The therapeutic efficacy is higher if cetuximab is combined with chemotherapy or radiotherapy, leading to a response rate of up to 23 %, which is still quite low [4,5]. Increasing evidence indicates that many patients develop resistance to cetuximab therapy [6]. Mutations in KRAS, EGFR, and BRAF or the activation of other receptor tyrosine kinases such as erythroblastosis oncogene B2 (erbB2) or ErbB3 limit the beneficial effects of cetuximab by bypassing EGFR signaling [6-8]. Moreover, tumor progression is frequently associated with alterations in DNA repair pathways [9]. This dysfunctional DNA repair capacity results in genomic instability of tumor cells [10]. Recent reports have demonstrated that defective DNA repair in tumor cells can be linked to acquired therapy resistance [11]. Therefore, novel agents that target DNA repair pathways in tumors are under investigation [12]. Interestingly, a recent study showed that treatment with cetuximab and the poly-ADP-ribose polymerase inhibitor ABT888 enhances cell death in comparison to cetuximab alone [13]. In the present study, we addressed the mechanism of acquired cetuximab resistance in several tumor entities. We found that cetuximab led to increased protein levels of the DNA repair–related endonuclease essential meiotic endonuclease 1 homolog 1 (Eme1) in tumor cell lines as well as in primary cultures of human glioblastoma cells [14]. Eme1 is a regulatory subunit of the heterodimeric endonuclease methyl methanesulfonate–sensitive UV-sensitive 81 (Mus81)/Eme1, which belongs to the xeroderma pigmentosum group F (XPF)/Mus81 protein family and acts by cleaving stalled or blocked replication forks, thereby promoting DNA repair and maintaining genomic integrity [14,15]. The elevated Eme1 protein levels resulted in increased DNA repair in the tumor cells. Moreover, cetuximab treatment protected these cells from apoptosis triggered by exposure to ultraviolet-C (UVC)

light or by megavoltage irradiation–induced DNA damage. This protective effect was dependent on elevated levels of Eme1. Taken together, these findings strongly suggest that Eme1 is a negative predictive marker for a combination therapy of cetuximab with treatment that induces DNA damage such as radiotherapy. Materials and Methods

Cell Culture and Drug Treatments A431 and A172 were obtained from the American Type Culture Collection (ATCC) (Manassas, VA); MDA-MB-231 cells were obtained from Prof Dr E. Dahl (Institute of Pathology, RWTH Aachen University, Aachen, Germany). All cell lines were cultured in Dulbecco′s modified Eagle’s medium (Gibco, Carlsbad, CA) with 10% FBS (BioWest, Nuaillé, France). For EGFR inhibition, concentrations of 50, 100, and 500 μg/ml of the monoclonal antibody cetuximab (Erbitux) were used; concentrations of up to 100 μg/ml have been used previously in experimental studies on cetuximab effects [6,16].

Glioblastoma Samples From October to December 2012, glioblastoma samples from 10 consecutive patients with a histologically proven supratentorial glioblastoma multiforme (GBM) (World Health Organization (WHO) grade IV) were included. Samples were provided by the local Department of Neurosurgery (RWTH Aachen University). Experiments were approved by the local ethics committee of the Medical Faculty, RWTH Aachen University (EK 220/12). Subjects gave written informed consent for their participation.

Culture of Primary Glioblastoma Cells Samples were processed within 2 hours after surgical removal. Tumor cells were washed and dissociated by mechanical and enzymatic means into single cells, resuspended in Dulbecco′s modified Eagle’s medium/20% FBS, and cultured on poly-L-Lysine (PLL) (Sigma-Aldrich, St Louis, MO)–coated plates. Medium was changed the next day to remove erythrocytes. Experiments were performed with cells from passage No. 2.

Radiotherapy Cells were irradiated with megavoltage X rays (linear accelerator; Siemens, Erlangen, Germany) or UVC (germicidal lamp, 200-280 nm; intensity = 135 J/cm 2 or 270 J/cm 2; OSRAM, München, Germany) 24 hours after addition of cetuximab. Radiotherapy was performed as a

Neoplasia Vol. 16, No. 3, 2014 6-MV single-shoot irradiation of 10 or 20 Gray (Gy) at the local Department of Radiation Oncology (RWTH Aachen University).

Cell Number Assay (Cell Counting) Cells were seeded onto culture dishes containing a grid (2 × 2 mm) and incubated without or with cetuximab for 72 hours. Cell numbers were determined by counting, and control (without cetuximab) was set as 100%.

Apoptosis/Necrosis Assay (FACS) Cells were detached from cell culture dish with Accutase (Invitrogen, Grand Island, NY). For detection of necrotic and apoptotic cells, Propidium Iodide (Sigma-Aldrich) and annexin V, Pacific Blue (Invitrogen) were added to Annexin buffer (10 mM Hepes, 140 mM NaCl, 2.5 mM CaCl2, pH 7.4). Cells were analyzed by flow cytometry using a FACSCanto II (BD Biosciences, Franklin Lakes, NJ, USA). Data were analyzed using FlowJo (Tree Star Inc., Ashland, OR, USA) software.

DNA Synthesis Assay (Bromodeoxyuridine) Cells were seeded onto PLL-coated glass coverslips and incubated for the indicated periods of time. Bromodeoxyuridine (5-bromo-2'deoxyuridine) (BrdU; 10μM, Sigma-Aldrich) was added to culture medium for 20 minutes before rinsing with phosphate-buffered saline and fixation with 4% paraformaldehyde (15 minutes). DNA was denatured with 2 N HCl for 15 minutes at 37°C. Cells were incubated with 0.1 M sodium borate (Sigma-Aldrich) (pH 8.5) for 10 minutes. Indirect immunofluorescence (IF) was carried out as described previously [17]. Cells were incubated with the primary antibody against BrdU (1:100) overnight and with Hoechst 33342 (1:10 000; Invitrogen) and secondary antibody (1:1000) for 1 hour. Images were analyzed using AxioVision Rel.4.6 (Carl Zeiss, Oberkochen, Germany) to determine the percentage of the BrdU-labeled cells.

Cell Viability (alamarBlue) Cells were seeded onto PLL-coated 24-well tissue culture dishes and 24 hours later transfected with small interfering (si)RNA and/ or incubated in the absence or presence of cetuximab for the indicated periods of time. alamarBlue (Invitrogen) assay was performed following the manufacturer’s instructions. To determine cellular metabolic activity, the fluorescence was monitored (wavelengths = 530 nm, excitation; 590 nm, emission) with a microplate reader (FLUOstar OPTIMA; BMG Labtech, Ortenberg, Germany). For analysis, viability mean value of cells grown in the absence of cetuximab (control) was set as 100%.

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used as a reporter to measure ubiquitin-proteasome–dependent degradation, were performed using Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer’s recommendations.

siRNA, siRNA Transfection, and Quantitative Real-Time Polymerase Chain Reaction Cells were transfected using HiPerFect (Qiagen) following manufacturer’s instructions. The efficiency of each transfection was monitored by quantitative real-time polymerase chain reaction (PCR) and Western blot (WB) analysis 48 hours after transfection. siRNA (siGENOME SMART pool) against human Eme1 (siEme1) and control nontargeting siRNA (siGENOME) (siC) were purchased from Dharmacon (Thermo Fisher Scientific, Lafayette, CO, USA). One microgram of cDNA was used for quantitative real-time PCR using SensiMix Plus SYBR Kit (Bioline, London, United Kingdom) following manufacturer’s recommendations and run on a Rotor-Gene 6000 (Corbett Life Science/Qiagen) machine. Mean mRNA levels of the gene of interest were calculated relative to the levels of ß-glucoronidase. For mRNA quantification, the following primer pairs were used: ß-glucoronidase, 5’CTCATTTGGAATTTTGCCGATT-3’ and 5’-CCGAGTGAAGATCCCCTTTTTA-3’; Eme1 primers were purchased from Qiagen (QT00079940).

Lysate Preparation and WB Analysis WB analysis was carried out as described previously [17]. The membrane was probed with the following first antibodies used: from Cell Signaling Technology (Danvers, MA, USA), (dilution 1:1,000) antiEGFR, anti-phospho (p)EGFR (Y1173), anti-pAkt (S473), anti-Akt, anti–phospho-extracellular signal-regulated kinase (anti-pErk) (T202/ 204), anti-Erk, anti–phospho-signal transducer and activator of transcription 3 (anti-pSTAT3) (Y705), anti-STAT3, anti–Bcl extra large (anti-BclXL), anti–B cell lymphoma 2 (Bcl2), anti–phosphocheckpoint kinase 1 (anti-pChk1) (S345), anti-pChk2 (T68), antipHistone H2A.X (S139), anti-p-p53 (S15), anti–phospho-breast cancer tumor suppressor 1 (anti-pBRCA1) (S1524), anti-pWee1 (S642), and anti-Wee1; from Abcam (Cambridge, England), anti-Eme1 (1:1000); from Sigma-Aldrich (St. Louis, MO, USA), anti–α-Tubulin (1:10,000); and from Santa Cruz (Santa Cruz, CA, USA) Biotechnology, antiUbiquitin (1:1000). As secondary antibodies (dilution 1:5,000), goat anti-mouse and goat anti-rabbit, both HRP conjugated (Thermo Scientific), were used. Immunoreactive proteins were detected by enhanced chemiluminescence (Amersham Biosciences, Piscataway, NJ). Densitometric quantification of the band intensity was normalized to α-Tubulin levels using Adobe Photoshop CS5 (San Jose, CA, USA).

Alkaline Comet Assay

Total RNA was isolated using the RNeasy Mini Kit (Qiagen, Valencia, CA) according to the manufacturer’s recommendations. Cell lysates were homogenized using QIAshredder (Qiagen) columns, and genomic DNA was digested by the RNAse-free DNase set (Qiagen) for 15 minutes at room temperature (RT). The concentration of RNA was determined using a Photometer (Eppendorf, Hamburg, Germany). cDNA was synthesized with the Reverse Transcription System (Promega, Fitchburg, WI). For cDNA synthesis, one microgram of RNA was applied.

To detect radiation-induced DNA damage, a Comet Assay kit (Cell Biolabs, Inc, San Diego, CA) was used according to the manufacturer’s instructions. Alkaline electrophoresis took place at 1 V/cm for 30 minutes at 4°C. The comet images were captured with Carl Zeiss Axiovert 200 M (Carl Zeiss, Oberkochen, Germany) inverted fluorescence microscope. The lengths of 30 comet tails were randomly measured per slide (distance measured from the center of the head to the end of the tail using AxioVision Rel.4.6).

Transient Transfection

Immunofluorescence

Transient transfections with d1EGFP (Clontech, Mountain View, CA, USA), which encodes an unstable EGFP that can be

Indirect IF was carried out as described previously [17]. The following first antibodies (dilution 1:100) were used: anti-Eme1

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Figure 1. Effects of cetuximab on EGFR signaling and cell proliferation. (A) A431 cells were treated with 100 μg/ml cetuximab for the indicated periods of time. Whole-cell lysates were analyzed for the expression and phosphorylation of the designated proteins by immunoblot analysis. (B) A431 cells were treated with the indicated amounts of cetuximab for 72 hours. The cells were then counted and compared to the control (set to 100%). The differences in cell number in response to cetuximab treatment were not statistically significant. The data represent mean values and SDs of three experiments. (F) A431 cells were treated with cetuximab for 48 hours. DNA synthesis was measured by incorporation of BrdU. That was added during the last 30 minutes before cells were fixed. The data represent mean values and SDs of three experiments. (C) A431 cells were incubated with the indicated amounts of cetuximab for 14 days. Metabolic activity was measured using the alamarBlue assay. The quantification is representative of three independent experiments. (D) Image was taken 14 days after addition of cetuximab (100 μg/ml) and of untreated A431 cells. (E) A 431 cells were treated for indicated periods of time. The expression of the indicated proteins was analyzed in whole-cell lysates by immunoblot analysis. #P N .05; *P b .05; and **P b .01.

and anti-BrdU. The following secondary antibodies (dilution 1:1000) were used: Alexa Fluor™ 488–conjugated anti-rabbit, Alexa Fluor™ 555–conjugated anti-mouse and Alexa Fluor™ 555– conjugated anti-rabbit (Invitrogen). Hoechst 33342 was obtained from Sigma-Aldrich. Images were taken with Carl Zeiss Axiovert 200 M inverted fluorescence microscope and equipped with an ApoTome (Carl Zeiss, Oberkochen, Germany) slider. Images were analyzed using AxioVision Rel.4.6.

Statistical Analysis Unpaired Student’s t test was used to evaluate significance between two sample groups. Values were expressed as means ± SD from three independent experiments. Differences were considered as statistically significant when P b .05. Error bars indicate the SD of triplicate measurement, (*) indicates significance in comparison to controls with (***) = P b .001, (**) = P b .01, and (*) = P b .05; (#) indicates no significant difference.



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Figure 2. Effects of cetuximab on the expression of the Eme1 endonuclease. (A) A431 cells were treated with 100 μg/ml cetuximab. Lysates were prepared at the indicated time points and subjected to WB analysis using the indicated antibodies. (B) Densitometric analysis of Eme1 from Figure 2A; the data represent mean values and SDs of three experiments. (C) A431 cells were incubated with 1, 10, or 100 μg/ml cetuximab for 1 and 72 hours. Cell lysates were analyzed by immunoblot analysis to detect Eme1 and α-Tubulin. (D) A431 cells were pretreated for 4 hours with 5 μM Stattic. Cetuximab was added for 1 hour. Lysates were subjected to WB analysis using the indicated antibodies.( E) A431 cells were left untreated or were incubated with 100 μg/ml cetuximab for 24 hours. Eme1 (red) was detected by indirect IF; Hoechst nuclear counterstain (blue); scale bar, 10 μm. (F) Fluorescence intensity of Eme1 was quantified in the nuclei of 20 cells per experiment. The graphs show mean values and SDs of three experiments; AU, arbitrary units.

Results

Cetuximab Inhibits Activation of EGFR, Akt, and Erk1/2 but Stimulates STAT3 Cetuximab prevents binding of ligands to the EGFR and thereby inhibits the subsequent activation of downstream signal transduction pathways [3]. A431 cells, which express high levels of the EGFR, show tyrosine phosphorylation of the receptor and strong Erk phosphorylation when grown in medium containing serum. In line with published results [18], we found that incubation of A431 cells with 100 μg/ml cetuximab reduced receptor phosphorylation and led to down-regulation and decreased activity of EGFR (Figure 1A). Moreover, this treatment resulted in decreased phosphorylation of the downstream signaling kinases Akt and Erk1/2 (Figure 1A). In contrast, increased STAT3

phosphorylation was observed (Figure 1A). Together, these findings suggested that cetuximab treatment affected distinct signaling pathways inversely.

Cetuximab Fails to Affect Cell Proliferation but Increases DNA Synthesis In several studies, incubation of A431 cells with cetuximab resulted in a decrease of cell numbers [19,20]. In these studies, cells were detached from the cell culture plates before the cell survival assay. In the current study, we confirmed that cetuximab treatment and subsequent detachment induced cell death (Figure W1A). However, we also observed that nondetached A431 cells did not show reduced cell proliferation or viability during incubation for 72 hours with cetuximab (Figures 1B and W1B). Only when A431 cells were incubated for 14 days with cetuximab (10 and

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Figure 3. Cetuximab treatment induces Eme1 protein stability. (A) Quantitative real-time PCR analysis of Eme1 target gene in A431 cells after treatment with or without cetuximab for 48 hours. Error bars represent SDs of biologic triplicates. (B) A431 cells were treated with 3μM MG132 for 4 hours; 100 μg/ml cetuximab was added during the last 2 hours. Whole-cell lysates were analyzed for the indicated proteins by immunoblot analysis. (C) Densitometric quantification of Eme1 from B; the data represent mean values and SDs of three experiments. (D) A431 were transfected either with green fluorescence protein (GFP) or d1EGFP plasmids; 24 hours after transfection, MG132 (3 μM) or cetuximab (100 μg/ml) was added for additional 24 hours. Cell lysates were analyzed for the indicated proteins by immunoblot analysis.

100 μg/ml) cell viability and growth were decreased by 10% (Figure 1, C and D). This was consistent with the constant expression of antiapoptotic proteins Bcl extra large (BclXL) and B cell lymphoma 2 (Bcl2) (Figure 1E). Instead, cetuximab was found to stimulate the incorporation of BrdU, suggesting that the cells responded with enhanced DNA synthesis (Figure 1F). Together, these findings indicated that cetuximab induces DNA synthesis in the absence of enhanced cell proliferation.

STAT3 Signaling Is Crucial for Cell Survival As shown above, treatment of A431 cells with cetuximab stimulated the phosphorylation of STAT3, a modification that is associated with enhanced activity (Figure 1A). Previous reports documented STAT3 as an important factor responsible for tumor cell survival and proliferation [21,22]. To address the role of STAT3 signaling for the survival of A431 cells on cetuximab treatment, we incubated these cells with the small-molecule STAT3 inhibitor Stattic (Sigma-Aldrich, St. Louis, MO, USA), which prevents dimerization and nuclear accumulation of the STAT3 protein [23]. The inhibition of STAT3 reduced cell viability in a dose-dependent manner within 24 hours (Figure W2), implying that STAT3 signaling is crucial for A431 cell viability.

Cetuximab Causes Up-Regulation of Eme1 DNA synthesis occurs during cell proliferation, when DNA is replicated [24]. However, on the basis of the results described above, the increase in DNA synthesis in the presence of cetuximab did not correlate with enhanced proliferation (Figure 1, B and C). DNA synthesis also occurs during DNA repair [25]. Moreover, recent studies described a functional link between STAT3 and Eme1/ Mus81, a heterodimeric endonuclease involved in DNA repair [26]. Therefore, we investigated the influence of cetuximab on the expression of Eme1, because this protein is the regulatory subunit of the functional endonuclease complex [27]. We found that incubation of A431 cells with 100 μg/ml cetuximab for up to 72 hours stimulated the expression of Eme1 (Figure 2A; quantification in Figure 2B). Moreover, even lower concentrations of cetuximab (1 and 10 μg/ml) led to elevated levels of Eme1 (Figure 2C ).

STAT3 Activation Leads to Elevated Levels of Eme1 To investigate if STAT3 inhibition blocks cetuximab-induced Eme1 up-regulation, we inhibited STAT3 activity with Stattic. Indeed, we found that Eme1 is not upregulated by cells incubated simultaneously with cetuximab and Stattic (Figure 2D).



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Figure 4. Eme1 knockdown eliminates cetuximab-mediated effects on DNA synthesis. (A) A431 cells were transiently transfected with control siRNA (siC) or Eme1-specific siRNA (siEme1). Forty-eight hours later, the expression levels of Eme1 were quantified by quantitative real-time PCR as described in Materials and Methods section. The quantification is representative of three independent experiments. (B) Eme1 expression levels 48 hours following knockdown were assessed by analyzing cell lysates by WB analysis. Eme1 could only be visualized by additional treatment with 3 μM MG132, which was added 2 hours before lysis; 100 μg/ml cetuximab was added for 24 hours. (C) A431 cells were transiently transfected for 72 hours with control or Eme1-specific siRNA; 24 hours after transfection, cetuximab (100 or 500 μg/ml) was added. DNA synthesis was measured by incorporation of BrdU (addition of BrdU during the last 30 minutes). The data represent mean values and SDs of three experiments. (D) A431 were treated with 100 μg/ml cetuximab for 1, 24, and 48 hours. Whole-cell lysates were analyzed for the expression and phosphorylation of the designated proteins by immunoblot analysis.

Additionally, we visualized the intracellular localization of Eme1 (Figure 2E). Whereas untreated cells showed a weak staining, prominent nuclear staining of Eme1 immunoreactivity was observed in response to cetuximab (Figure 2E and quantification in Figure 2F). This suggested that cetuximab led to enhanced levels of Eme1 in A431 cells, which might be associated with enhanced DNA synthesis and repair.

glutamate, serine, and threonine sequence at its C terminus and has a half-life of ~ 1 hour [28,29]. We compared the effect of cetuximab on Eme1 and d1EGFP. The latter was not stabilized by cetuximab, arguing that it did not result in a generalized inhibition of ubiquitinproteasome–dependent protein degradation (Figure 3D). We concluded that cetuximab interfered with the stability of Eme1 by altering its availability to proteasomal degradation (Figure 3, B–D).

Cetuximab Promotes Eme1 Stability

Eme1 Mediates Induction of DNA Synthesis

To further evaluate how cetuximab treatment stimulated Eme1 protein expression, we addressed whether cetuximab affected the transcription or the stability of the protein. Therefore, we investigated mRNA levels of Eme1 in cetuximab-treated and untreated cells. We did not observe a significant alteration of Eme1 mRNA expression in response to cetuximab (Figure 3A). However, blocking protein degradation with the proteasomal inhibitor MG132 (Sigma-Aldrich, St. Louis, MO, USA) enhanced Eme1 protein expression, suggesting that the levels of this protein might be regulated by the ubiquitin-proteasomal system (Figure 3B and quantification in Figure 3C). To confirm this hypothesis, we transfected cetuximab-treated A431 cells with a model substrate of the proteasome (d1EGFP) (Figure 3D). d1EGFP is a destabilized enhanced green fluorescence protein, which contains a proline,

The induction of DNA synthesis and Eme1 expression suggested that cetuximab treatment promotes an Eme1-dependent increase in DNA repair. Hence, we investigated whether Eme1 is responsible for the increased DNA synthesis during incubation with cetuximab (Figure 4). We suppressed Eme1 in A431 cells using Eme1-specific siRNAs (siEme1). At the mRNA level, a reduction of 75% was achieved 48 hours after transfection (Figure 4A). At the protein level, a reduction of 80% was obtained (Figure 4B). Subsequently, we investigated the influence of cetuximab on DNA synthesis in Eme1expressing and Eme1 knockdown cells. BrdU incorporation demonstrated that the Eme1 knockdown blocked the increase in DNA synthesis induced by cetuximab, whereas the transfection of a control siRNA (siC) was unable to inhibit the cetuximab-dependent increase in BrdU incorporation (Figure 4C). Furthermore, we

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Figure 5. Eme1 mediates cetuximab-induced DNA repair. (A and B), DNA repair was determined by comet assay. (A) Untreated or cetuximab (100 μg/ml for 24 hours)-treated A431 cells were exposed to UVC light (135 J/cm 2). Comet assays were carried out immediately after irradiation (UVC) or after the cells had recovered for 2 hours at 37°C (UVC (2h)). For each experiment, comet tail lengths of 30 cells were determined. The data represent mean values and SDs of three experiments. (B) A431 cells were transiently transfected with control siRNA (siCon) or Eme1-specific siRNA to knockdown Eme1 (siEme1). Forty-eight hours later, 100 μg/ml cetuximab was added. Comet assays were carried out following 2-hour incubation at 37°C. The analysis was performed as described above. Mean values and SDs of three experiments. (C) A431 cells were treated with the indicated amounts of cetuximab for 24 hours. Following irradiation with UVC light (270 J/cm 2), cells were incubated in the absence or presence of cetuximab for another 48 hours. Metabolic activity was measured using an alamarBlue assay. The quantification is representative of three independent experiments. (D) As in C except that the cells were irradiated with 20 Gy instead of UVC treatment and following irradiation, cells were incubated for 6 days. The quantification is representative of three independent experiments.

visualized the pattern of Eme1 in BrdU-positive cetuximab-treated cells using indirect IF (Figure W3). After cetuximab treatment, we found as expected a prominent Eme1 staining especially in cell nuclei incorporating BrdU. Moreover, we detected an increased nuclear colocalization of BrdU and Eme1. These findings suggest that Eme1 is important for the cetuximab-mediated increase in DNA synthesis.

Cetuximab Triggers DNA Repair through Eme1 To expand on the observations described above, we addressed whether cetuximab was able to regulate DNA repair. The DNA damage response (DDR) can be initiated through various signaling pathways resulting in the activation of distinct DNA repair processes. Especially the function of the Chk1 was important to us, because it has been demonstrated that Chk1 influences the activity of the Mus81/Eme1 endonuclease [30]. Moreover, STAT3 promotes the DDR and seems to be important for Chk1 activity [31]. Consistent with this, we observed that in cetuximab-treated cells, the phosphorylation of Chk1 at serine 296 was elevated (Figure 4D and quantification in Figure W4). Subsequently, we analyzed the phosphorylation of additional proteins involved in the DDR. We found that already the treatment with cetuximab for 1 hour

stimulated the phosphorylation of the Chk2 at threonine 68, a modification that is associated with activity, and Histone H2A.X serine 139 phosphorylation. The phosphorylation of p53 at serine 15 was elevated after 24 and 48 hours. However, cetuximab did not alter the phosphorylation of the BRCA1 (Figure 4D and quantification in Figure W4). Together, these observations are consistent with stimulation of DNA repair. To visualize the cetuximab-mediated DNA repair, we next induced DNA damage in A431 cells using UVC light. UVC exposure creates UV-specific base alterations such as cyclobutane pyrimidine dimers and (6-4) photoproducts leading to DNA double-strand breaks (DSBs) during replication [32,33]. On DNA damage, short DNA fragments accumulate in the nucleus, which can be visualized by the comet assay (Figure W5). This assay was performed on cetuximabtreated and untreated cells immediately after UVC exposure and on cells that were incubated for two additional hours at 37°C (Figures 5A and W5). We observed that UVC light induced DNA damage to the same extent in untreated and in cetuximab-treated cells. However, the comet tail of cetuximab-treated cells was significantly shorter when cells were incubated for two additional hours. This indicated that, within these 2 hours, DNA repair took place, reducing the number of



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Figure 6. Eme1 is responsible for survival of UVC-treated cells. A431 cells were transiently transfected with control siRNA or Eme1specific siRNA. Following incubation for 24 hours, 100 or 500 μg/ml cetuximab was added. Thereafter, cells were irradiated with UVC light (135 J/cm 2) and incubated for another 24 hours at 37°C. Metabolic cell activity was measured as in Figure 5C. The data of three experiments were quantified, and mean values and SDs are shown.

DNA fragments that migrated out of the nucleus, whereas in control cells, no obvious DNA repair occurred (Figures 5A and W5). Next, we investigated whether cetuximab-induced DNA repair was dependent on Eme1. Therefore, we knocked down Eme1 in A431 cells and analyzed the DNA fragmentation in response to UVC treatment. Eme1 knockdown cells failed to induce DNA repair on cetuximab treatment (Figure 5B). From this, we concluded that Eme1 was critically involved in cetuximab-mediated DNA repair.

control cells (Figure 6). Still, a slight increase in survival of UVCtreated Eme1 knockdown cells was measurable in response to cetuximab, which might be due to residual Eme1 expression and/or to a small number of untransfected cells. Taken together, these findings support the notion that UVC-exposed cetuximab-treated cells escape death through Eme1-dependent DNA repair.

Cetuximab Induces Cell Survival after Irradiation

A431 cells are protected from UVC-induced DNA damage by cetuximab-dependent up-regulation of Eme1. To expand our observations to other tumor cells, we analyzed the effect of cetuximab on cell lines derived from tumor entities, which are targets of cetuximab therapy [35,36]. In the breast cancer cell line MDA-MB231 and the glioblastoma cell line A172, increased Eme1 protein levels and BrdU incorporation were measurable on cetuximab treatment (Figures 7, A–C, and W6). Cetuximab also promoted survival of these cells after treatment with DNA-damaging agents, including UVC light and megavoltage irradiation (Figure 7, D–G). The effect of cetuximab was dose dependent; more cells survived with higher concentrations of cetuximab, similar to the findings obtained for A431 cells.

The combination of radiotherapy with cetuximab in colorectal cancer treatment results in reduced response rates. Radiation induces cell death by DNA damage [34]. As we were able to demonstrate that cetuximab stimulated DNA repair at least in part by increasing the levels of the endonuclease Eme1, we investigated whether these cells were more resistant to DNA damage–induced cell death. Therefore, we treated A431 cells with UVC light in the presence or absence of cetuximab and measured metabolic cell activity 24 hours later. Indeed, cetuximab protected the UVC light–treated cells from death (Figure 5C). The effect of cetuximab was dose dependent; the largest effect on cell survival was achieved with the highest concentration of cetuximab. Hence, we concluded that after UVC light–induced DNA damage, cetuximab protected these cells from cell death due to Eme1-mediated DNA repair. To address whether this effect was specific for UVC treatment, we investigated the consequences of megavoltage irradiation on cetuximabtreated cells (Figure 5D). Cetuximab treatment of the irradiated A431 cells also led to significantly increased cell survival (Figure 5D), even though the cell death was less dramatic under the experimental conditions used compared to UV treatment.

Eme1 Is Responsible for DNA Repair and Survival of Cetuximab-Treated Cells We then analyzed whether the knockdown of Eme1 sensitized A431 cells to UVC light. In line with the above-described results, we observed that metabolic cell activity was decreased significantly in UVC-treated cells, in which Eme1 was knocked down compared to

Cetuximab Triggers Eme1-Mediated DNA Repair in Other Tumor Cell Lines

Cetuximab-Mediated Increase of Eme1 Protein in Primary Glioblastoma Cells Next, we investigated the effects of cetuximab on Eme1 in cultures of primary glioblastomas from a cohort of 10 tumor patients (Figure 8). In 5 of 10 cases, cetuximab caused elevated levels of Eme1 (Figure 8A). We detected a prominent EGFR expression in four of these five cases. In cetuximab-treated cultures of cells from four glioblastomas in which Eme1 was increased, we found increased STAT3 phosphorylation. These findings support our hypothesis that STAT3 activation results in elevated Eme1 expression in response to cetuximab application. The intracellular localization of Eme1 was visualized in one of the primary tumor cells (GBM1). Untreated cells showed predominantly

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Figure 7. Cetuximab triggers DNA repair in breast cancer and glioblastoma cell lines. (A) MDA-MB-231 (breast cancer) and A172 (GBM) cells were incubated with 100 μg/ml cetuximab for the indicated periods of time. Cell lysates were analyzed for Eme1 and α-Tubulin by immunoblot analysis. (B and C) MDA-MB-231 (B) or A172 (C) cells were treated with the indicated amounts of cetuximab for 48 hours. DNA synthesis was determined by measuring incorporation of BrdU. BrdU was added during the last 30 minutes before cell harvesting. The mean values and SDs of three experiments are illustrated. (D–G) MDA-MB-231 (D and E) or A172 (F and G) cells were treated with cetuximab for 24 hours as indicated. Following irradiation with UVC light (MDA-MB-231 cells with 135 J/cm 2 and A172 cells with 270 J/cm 2) or megavoltage irradiation (MDA-MB-231 cells with 10 Gy and A172 cells with 20 Gy), cells were incubated in the presence or absence of cetuximab for another 48 hours (UVC) or 6 days (megavoltage irradiation). Metabolic cell activity was measured using the alamarBlue assay. The quantification is representative of three independent experiments.

cytoplasmic and only weak nuclear Eme1 staining. In response to cetuximab, the overall staining was enhanced, and in particular, stronger nuclear Eme1-specific signals were measured (Figure 8B).

Additionally, DNA damage was induced in GBM1 cells by UVC irradiation. As expected, an increased viability of the cetuximabtreated cells was detected 24 hours after irradiation (Figure 8C). This



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Figure 8. Cetuximab treatment leads to elevated levels of Eme1 in primary brain cultures. (A) Primary glioblastoma cells from 10 patients were treated with 100 μg/ml cetuximab for 24 hours. Whole-cell lysates were analyzed for the indicated proteins by immunoblot analysis. Cetuximab treatment increased Eme1 protein levels in five primary glioblastomas. (B) Primary glioblastoma cells of one patient (GBM1) were incubated with 100 μg/ml cetuximab for 24 hours. Eme1 (red) was detected by indirect IF; Hoechst nuclear counterstain (blue). (C) Cells from patient GBM1 were incubated without or with 100 μg/ml cetuximab for 24 hours. Following irradiation with UVC light (J/cm 2), cells were incubated in the absence or presence of cetuximab for another 48 hours. Metabolic activity was measured using an alamarBlue assay. The quantification is representative of three independent experiments.

indicated that at least a subpopulation of primary glioblastoma cells responded to cetuximab by enhanced Eme1 protein levels and increased survival after irradiation. Discussion The EGFR is highly activated in many tumors [37]. The monoclonal antibody cetuximab is designed to inhibit EGFR-ligand interaction and the subsequent activation of downstream factors, which control cell survival and proliferation [3]. As a consequence of this molecular targeted therapy, tumor cells should cease proliferation and undergo

apoptosis, as shown in a variety of tumor cell lines [38]. However, the therapeutic efficacy of cetuximab in tumor therapy is only moderate [4]. Some patients fail to show any response to cetuximab, whereas others develop resistance later on during cetuximab treatment [6,8]. In the present study, we found that treatment of A431 cells with cetuximab decreased EGFR phosphorylation and activation of the signaling kinases Akt and Erk1/2 (Figure 1). However, we did not detect changes in survival or cell proliferation of nondetached cells, probably because cetuximab led to elevated activation of STAT3 in these cells (Figure 1). Lu et al. have shown previously that treatment of A431 cells

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with cetuximab increases STAT3 phosphorylation [39]. STAT3 is an important transcription factor involved in the promotion of cell survival and proliferation [21]. We found that the inhibition of STAT3 was sufficient to cause death of A431 cells (Figure W2), confirming reports on effects of STAT3 inhibition in other tumor cell systems [40]. It is unclear at present why cetuximab treatment of these cells leads to an increase in the phosphorylation of STAT3. Diverse mechanisms might contribute to the increase of activated STAT3 during EGFR inhibition. For example, Wu et al. demonstrated that the tyrosine kinase inhibitor gefitinib promotes STAT3 activation by downregulating STAT3 inhibitors such as suppressor of cytokine signaling 1 (SOCS1) and SOCS3 [41]. Another potential mechanism is mediated through the elevated nuclear EGFR levels in cetuximab-treated cells in vitro [42]. Lo et al. demonstrated that nuclear EGFR interacts physically and functionally with STAT3 [43]. It is thus possible that elevated nuclear EGFR binds to STAT3 leading to STAT3 activation. In this scenario, the inhibition of EGFR and its downstream signaling pathways by cetuximab would be reconciled with the simultaneous activation of STAT3. Moreover, the cross talk between Akt, Erk1/2 and STAT3 is altered by cetuximab; for example, the inhibition of Akt and Erk1/2 may lead to increased activation of factors controlling STAT3 signaling. It has been reported that increased STAT3 activation is associated with acquired resistance to cetuximab therapy [44]. Furthermore, our investigations demonstrate that cetuximabmediated activation of STAT3 promotes elevated levels of the regulatory subunit Eme1 of the heterodimeric endonuclease Mus81/ Eme1 in several tumor cell lines as well as in primary glioblastoma cells (Figures 2, 7, and 8). Eme1 together with its binding partner Mus81 forms a structure-specific 3’-flap DNA endonuclease and cleaves blocked or stalled replication forks [14]. The Mus81/Eme1 complex is involved in DNA repair pathways that remove UV light– induced DNA lesions and cross-links between DNA strands [45]. Recently, several reports indicated that a deficiency in DNA repair can play a role in acquired cetuximab resistance. For example, polyADP-ribose polymerase inhibitors have been investigated in tumors, which are defective in homologous recombination (HR)–mediated DNA DSB repair [13]. Nowsheen et al. found that additional treatment of different cancer cell lines with the poly-ADP-ribose polymerase inhibitor ABT-888 in combination with cetuximab enhanced cytotoxicity [13]. However, our findings demonstrate that cetuximab leads to a stabilization of the DNA repair-specific endonuclease Eme1, with the consequence of elevated Eme1 protein levels (Figure 3). We observed that the increased Eme1 levels in cetuximab-treated cells triggered DNA repair (Figures 1C and 5A). Only when Eme1 levels were reduced by siRNA treatment (Figure 4), DNA repair could be prevented in cetuximab-treated cells (Figure 5B). Furthermore, we investigated whether cetuximab induces changes in DDR pathways [46]. We found that cetuximab treatment increased the phosphorylation of the checkpoint kinases Chk1 and Chk2 and its downstream effectors p53 and Histone H2A.X (Figure 4D). Previous findings indicated a connection between the Eme1/Mus81-activity and the DDR system [47]. In fission yeast, for example, the cellular localization of Mus81/Eme1 is regulated by Cds1, a functional ortholog of human Chk1 [30]. As demonstrated above, cetuximab-mediated STAT3 activation regulates Eme1 levels. Wee1, a dual-specificity kinase, might also be involved in the regulation of Eme1-Mus81 activity. Martin et al. proposed a novel mechanism by which proteasomal degradation of Wee1 during S and G2/M phases of the cell cycle activates the

Neoplasia Vol. 16, No. 3, 2014 Eme1-Mus81 complex with subsequent cleavage of various substrates [42,43]. Indeed, we found that cetuximab treatment leads to a decrease in Wee1 total protein levels and Wee1 phosphorylation in A431 cells (Figure W7). These observations suggest that Wee1 could be a target of cetuximab and that it is activated upstream of Eme1. In this scenario, cetuximab might inhibit Wee1 phosphorylation, thereby resulting in the subsequent activation of the Eme1/Mus81 complex. Under these conditions degradation of Eme1 would not be initiated and thus Eme1 stabilized. As already mentioned, we observed that Eme1 triggers DNA repair in cetuximab-treated cells (Figure 5, A and B). DNA repair plays a central role in the response of cells treated with DNA-damaging agents such as UVC light or megavoltage irradiation. Our investigations demonstrate that cetuximab treatment protected several cancer cell lines from cell death following irradiation with UVC light or megavoltage X rays (Figures 5, C and D, and 7, D–G). Cetuximab prevented cell death by inducing DNA repair through Eme1 (Figures 5, A and B, and 6). In the clinical setting, cetuximab is often used in combination with radio- and/or chemotherapy because the efficacy of a cetuximab mono-therapy is quite low. However, the results from clinical studies that tested cetuximab in combination with radiochemotherapy differ widely. Some clinical trials support the use of cetuximab in combination with irradiation. Other studies failed to demonstrate any considerable improvement of progression-free survival of the patients or could only demonstrate a moderate increase in overall survival of 2 to 3 months [4,5]. The mechanisms underlying the failure of such combination therapies are widely discussed [48,49]. Ryu et al. proposed that the localization of EGFR plays a crucial role developing a combined cetuximab and radiotherapy resistance. However, Huang et al. reported that the loss of p53 that frequently occurs during tumor progression reduces cell sensitivity to both EGFR inhibitors and radiation [48,49]. Tomoda et al. demonstrated a link between elevated Eme1 expression and cisplatin resistance [50]. Cisplatin leads to the formation of interstrand cross-links [51]. Such cross-links are repaired by HR involving Eme1/Mus81 [52]. Short wavelength UV radiation (UVC) and ionizing radiation induce different kinds of DNA damage, but with a similar outcome, i.e., the formation of DSBs [53,54]. DSBs are a very toxic form of DNA lesion because both DNA strands are damaged and the only way to repair DSBs error-free is by using the complementary DNA strand as a template. This HR repair is one of the major repair mechanisms of DSBs [14,55]. The heterodimeric endonuclease Eme1 together with Mus81 plays an essential role in HR repair. The present findings support the notion that Eme1 is responsible for the development of radiotherapy resistance with increased Eme1 protein levels been measurable in cetuximab-treated cells (Figures 2A and 7A). Moreover, recent findings reported by Barry et al. suggest that STAT3 is important for efficient DNA repair because it modulates the DDR pathway [31]. Our results show that cetuximab induces the activation of STAT3 and DDR proteins (Figures 1A and 4D). In conclusion, previous clinical studies showed contradictory outcomes when cetuximab is used in combination with radiotherapy [5,56]. We propose that the therapy may fail when cetuximab leads to increased Eme1 protein levels. These elevated levels of the Mus81/ Eme1 endonuclease, which appear during short-term treatments with cetuximab, promote error-free DNA repair of DSBs in the irradiation paradigm, resulting in increased survival of the tumor cells. We expect that the identification of Eme1 as a negative predictive marker for


cetuximab therapy resistance will contribute to the development of new, molecular targeted therapies, in which cetuximab and radiotherapy could be combined with drugs that inhibit DNA repair pathways.

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Acknowledgments We thank E. Dahl, Institute of Pathology, RWTH Aachen University, for supplying the MDA-MB-231 cells. We are grateful to J. M. Lüscher-Firzlaff, Institute of Biochemistry and Molecular Biology, RWTH Aachen University, for her kind help and advice regarding the knockdown experiments. We thank J. Floehr, Institute of Biomedical Engineering, Biointerface Group, RWTH Aachen University, for help with the alamarBlue Assay and G. Brook, Institute of Neuropathology, RWTH Aachen University, for reading the manuscript.

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Supplementary materials

Figure W1. Detachment of cetuximab-treated A431 cells induces cell death. (A) A431 cells were incubated without or with (100 μg/ml) cetuximab for 1, 3, 24, or 48 hours. Cells were detached from cell culture plates and incubated with annexin V and Propidium Iodide to determine cetuximab-mediated cell death. The data represent mean values and SDs of three experiments. The amount of living cells was determined through comparison of cetuximab-treated with untreated cells; ***P b .001. (B) A431 cells were treated with the indicated amounts of cetuximab for 72 hours. Metabolic activity was measured using an alamarBlue assay. The quantification is representative of three independent experiments; #P N .05.

Figure W2. Effect of STAT3 inhibition on A431 cells. Exponentially proliferating A431 cells were incubated with the indicated increasing amounts of Stattic for 24 hours. Cell viability was determined using alamarBlue that measures the reducing capacity of cells. Displayed are mean values and SDs of three independent experiments; ***P b .001.

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Figure W3. Effect of cetuximab on Eme1 localization in BrdU-positive cells. A431 cells were incubated without or with cetuximab (100 μg/ml) for 48 hours. BrdU was added 30 minutes before cell fixation. Eme1 (green) and BrdU (red) were detected by indirect IF; scale bar, 10μm.

Figure W4. Effect of cetuximab on activation of DDR. Densitometric analysis of pChk1, pChk2, p-p53, pBRCA1, and pHistone H2A.X from three experiments was carried out.



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Figure W5. Influence of cetuximab on DNA repair visualized by comet assay. A431 cells were treated with 100 μg/ml cetuximab for 24 hours (C, D, G, and H), irradiated with UVC light (135 J/cm 2), and incubated for additional 2 hours at 37°C (E–H). For control cells, they were left untreated (A and B). DNA repair was determined using a comet assay.

Figure W6. Eme1 levels in MDA-MB-231 and A172 cells. Densitometric analysis of Eme1 protein levels as shown in Figure 6A. The data represent mean values and SDs of three experiments. (A) MDA-MB-231 cells; (B) A172 cells.

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Figure W7. Effect of cetuximab on Wee1. (A) A431 cells were treated as shown in Figure 1D. The cell lysates were analyzed for the indicated proteins by immunoblot analysis. (B and C) Densitometric analysis of phosporylated Wee1 (B) or total Wee1 (C); the data represent mean values and SDs of three experiments.