Disclosure of erlotinib as a multikinase inhibitor in pancreatic ductal adenocarcinoma

Abstract

A placebo-controlled phase 3 trial demonstrated that the epidermal growth factor receptor (EGFR) inhibitor erlotinib in combination with gemcitabine was especially efficient in a pancreatic ductal adenocarcinoma (PDAC) subgroup of patients developing skin toxicity. However, EGFR expression was not predictive for response, and markers to characterize an erlotinib-responding PDAC group are currently missing. In this work, we observed high erlotinib IC50 values in a panel of human and murine PDAC cell lines. Using EGFR small interfering RNA, we detected that the erlotinib response was marginally influenced by EGFR. To find novel EGFR targets, we used an unbiased chemical proteomics approach for target identification and quality-controlled target affinity determination combined with quantitative mass spectrometry based on stable isotope labeling by amino acids in cell culture. In contrast to gefitinib, we observed a broad target profile of erlotinib in PDAC cells by quantitative proteomics. Six protein kinases bind to erlotinib with similar or higher affinity (K(d) = 0.09-0.358 µM) than the EGFR (K(d) 0.434 µM). We provide evidence that one of the novel erlotinib targets, ARG, contributes in part to the erlotinib response in a PDAC cell line. Our data show that erlotinib is a multikinase inhibitor, which can act independent of EGFR in PDAC. These findings may help to monitor future erlotinib trials in the clinic.

Figures

Figure 1

Erlotinib IC50 values of murine and human PDAC cells. (A) Viability of PDAC cells after treatment with 6, 12, and 18 µM erlotinib was measured by MTT assay and compared to vehicle-treated controls. IC50 values of murine (black) and human (red) PDAC cell lines were calculated using a nonlinear regression model. Statistics can be found in Table 1. (B) Mean IC50 values of murine (black triangle) and human (red square) PDAC cell lines show no statistically significant differences (Student's t test, P = .15). (C) Western blot analysis of EGFR expression levels in the indicated PDAC cell lines. β-Actin controls equal protein loading. (D) Correlation of the erlotinib IC50 values with EGFR protein expression levels in 22 PDAC cell lines. The Spearman correlation coefficient and the P value (two-tailed) are indicated.

Figure 2

Erlotinib response of PDAC cells is EGFR independent. MiaPaCa2 (A), BxPc3 (B), T3M4 (C), and PPT6554 (D) cells were transfected with a control siRNA or an EGFR-specific siRNA. Left panel: Knockdown of the EGFR 48 hours after the transfection by Western blot analysis. β-Actin controls equal protein loading. Twenty-four hours after transfection, cells were treated with increasing doses of erlotinib as indicated for 48 hours or were left as an untreated control. Viability was determined using MTT assays. Middle panel: Viability of control siRNA-transfected cells was arbitrarily set to 100% and compared to (I) control siRNA-transfected and erlotinib-treated cells, to (II) EGFR siRNA-transfected cells, and to (III) EGFR siRNA-transfected and erlotinib-treated cells. Right panel: Erlotinib-induced therapeutic response in control and EGFR siRNA-transfected cells. Note that the right panels rely on an extrapolation of the data presented in the middle panels to better visualize the erlotinib-induced loss of viability (=therapeutic response) in control and EGFR siRNA-transfected cells. Student's t test, *P < .05 versus controls.

Figure 3

Target profiles of gefitinib and erlotinib. Shown are the target proteins of erlotinib (A) and gefitinib (B) in dispersed primary murine pancreatic cancer cells PPT6554. Kd values are plotted on a logarithmic scale, and target proteins are ranked from low (top) to high (bottom) Kd values. Protein kinases are depicted in blue; other kinases, purple; associated proteins, orange; and other proteins, green. Target proteins with Kd values lower than 10 µM are shown. In addition, target proteins with Kd values greater than 10 µM are indicated if they were identified as targets for both compounds. A complete list of all target proteins is provided in Table W2.

Figure 4

Role of ARG in the erlotinib response of PDAC cells. MiaPaCa2 (A), BxPc3 (B), T3M4 (C), and PPT6554 (D) cells were transfected with a control siRNA or an ARG-specific siRNA. Left panel: Knockdown of ARG 48 hours after the transfection by Western blot analysis. β-Actin controls equal protein loading. Twenty-four hours after transfection, cells were treated with increasing doses of erlotinib as indicated for 48 hours or were left as an untreated control. Viability was determined using MTT assays. Middle panel: Viability of control siRNA-transfected cells was arbitrarily set to 100% and compared to (I) control siRNA-transfected and erlotinib-treated cells, to (II) ARG siRNA-transfected cells, and to (III) ARG siRNA-transfected and erlotinib-treated cells. Right panel: Erlotinib-induced therapeutic response in control and ARG siRNA-transfected cells. Note that the right panels rely on an extrapolation of the data presented in the middle panels to better visualize the erlotinib-induced loss of viability (=therapeutic response) in control and ARG siRNA-transfected cells. Student's t test, *P < .05 versus controls.

Figure 5

Tyrosine phosphorylation of ARG is reduced on erlotinib treatment. (A) T3M4 cells were treated with erlotinib as indicated or were left as an untreated control. After 20 minutes, whole-cell extracts were prepared, and ARG was immunoprecipitated. Pre-immune IgGs were used as precipitation controls. Western blots were performed with ARG and anti-phosphotyrosine antibodies. The input (5%) was probed with ARG and anti-phosphotyrosine antibodies. (B) Western blot analysis of ARG protein expression levels in the indicated PDAC cell lines. β-Actin controls equal protein loading. (C) Correlation of the erlotinib IC50 values with the ARG protein expression in 22 PDAC cell lines. The Spearman correlation coefficient and the P value (two-tailed) are indicated. T3M4 cells are depicted as a red box. MiaPaCa2 (D), BxPc3 (E), T3M4 (F), and PPT6554 (G) were treated with imatinib as indicated or with vehicle as control. Viability was determined after 48 hours using MTT assays. Imatinib IC50 values and the 95% confidence interval (95% CI) are indicated.