1. Volume 16, Issue 3, Pages 207-220.e4 (March 2014)
Cetuximab Induces Eme1-Mediated DNA Repair: a Novel Mechanism for Cetuximab Resistance 
Agnieszka Weinandy, Marc D. Piroth, Anand Goswami, Kay Nolte, Bernd Sellhaus, Jose Gerardo-Nava, Michael Eble, Stefan Weinandy, Christian Cornelissen, Hans Clusmann, Bernhard Lüscher, Joachim Weis 
Neoplasia 
Volume 16, Issue 3, Pages e4 (March 2014)
DOI: /j.neo
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2. 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 > .05; *P < .05; and **P < .01.
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3. 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.
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4. 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.
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5. 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.
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6. 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/cm2). 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/cm2), 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.
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7. Figure 6
Eme1 is responsible for survival of UVC-treated cells. A431 cells were transiently transfected with control siRNA or Eme1-specific siRNA. Following incubation for 24 hours, 100 or 500 μg/ml cetuximab was added. Thereafter, cells were irradiated with UVC light (135 J/cm2) 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.
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8. 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/cm2 and A172 cells with 270 J/cm2) 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.
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9. 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/cm2), 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.
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10. 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) 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 > .05.
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11. 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 < .001.
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12. 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.
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13. 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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14. 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/cm2), 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.
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15. 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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16. 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.
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