Combination of cetuximab with chemoradiation, trastuzumab or MAPK inhibitors: mechanisms of sensitisation of cervical cancer cells

Abstract

Background: Cervical cancer (CC) annually kills 288,000 women worldwide. Unfortunately, responses to chemoradiation are partial and are of short duration. As anti-EGFR monoclonal antibodies sensitise tumours, we investigated cetuximab's toxicity plus chemoradiation on CC cells, which express different EGFR levels.

Methods: EGFR, HER2, AKT and MAPK expression and phosphorylation status were determined by western blotting. Cytotoxicity was assessed by MTT or clonogenic assays (CA) in cell lines treated with cetuximab alone or in combinations.

Results: Cetuximab with cisplatin and radiation achieved maximum cytotoxic effects for A431, Caski and C33A cells (high, intermediate and low EGFR expression, respectively) in CA. Cetuximab efficiently decreased MAPK and AKT phosphorylation in A431 cells but slightly less in Caski and C33A cells. To check whether further EGFR, HER2 or MAPK inhibition would improve cetuximab's cytotoxicity, we combined it with an EGFR tyrosine kinase inhibitor (TKI), trastuzumab or a MEK1/2 inhibitor (PD98059). In Caski, but not in C33A cells, cetuximab cooperated with the TKI, reducing cell survival and AKT and MAPK phosphorylation. However, cetuximab with trastuzumab or PD98059 reduced survival and MAPK phosphorylation of both cell lines.

Conclusion: Our data suggest that cetuximab combined with chemoradiation, trastuzumab or MAPK inhibitors has useful applications for CC treatment, independently of EGFR expression.

Figure 1

Effect of RxT, cisplatin or cetuximab treatments on the proliferation and cell-cycle distribution of A431, Caski and C33A cells. (A) Effects of different doses of RxT in clonogenic assays (CA). (B) Effects of different concentrations of cisplatin in MTT assay. (C) Effects of cetuximab (100 μg ml⁻¹) in CA. (D) Effects of cetuximab treatment in MTT assay. (E) Effects of cetuximab treatment (100 μg ml⁻¹) on cell-cycle phase distribution analysis by flow cytometry. (F) Induction of apoptotic bodies by cetuximab treatment (100 μg ml⁻¹) visualised by Hoechst staining. Student's t-test ^*P<0.05, compared to controls.

Figure 2

Effects of cetuximab (100 μg ml⁻¹) alone or combined with cisplatin (1.0, 0.5 and 0.15 μM for A431, Caski and C33A cells, respectively) and/or RxT (2.0, 1.5 and 0.3 Gy for A431, Caski and C33A cells, respectively) on survival in CA. (A) A431, (B) Caski and (C) C33A cells. (D) Representative picture of A431 cells under different treatments as described in material and methods. One-way analysis of variance (ANOVA) with Tukey's post test ^*P<0.05, when compared to control cells.

Figure 3

Western blotting analysis of basal and phosphorylated signalling pathways before and after cetuximab (100 μg ml⁻¹) treatment. (A) Basal levels of total and phosphorylated EGFR, HER2/neu and downstream signalling proteins. (B, C and D) Effects of cetuximab on EGF-induced activation of EGFR (Tyr 845, 992, 1045 and 1068), HER-2/neu, AKT and ERK1/2 of A431, Caski and C33A cells, respectively, detected by western blotting.

Figure 4

Effects of cetuximab (100 μg ml⁻¹) alone or combined with trastuzumab (10 μg ml⁻¹), an anti-HER2 MAb, or PD153035 (0.1 μM), an EGFR tyrosine kinase inhibitor, or PD98059 (25 μM), a MAPK pathway inhibitor, on Caski and C33A cells. (A) Effects of cetuximab alone or combined to trastuzumab on the survival in CA. (B) Representative pictures of Caski and C33A cells under cetuximab and trastuzumab treatments in CA. (C and D) Western blotting analysis of the inhibition of EGF-induced phosphorylation of AKT and p44/42 (ERK1/2) by cetuximab alone or combined with trastuzumab in Caski and C33A cells, respectively. (E) Effects of cetuximab alone or combined with PD153035 on cell survival in CA. (F) Effects of cetuximab alone or combined with PD98059 on proliferation by MTT assays. (G) Western blotting analysis of the inhibition of EGF-induced phosphorylation of EGFR (Tyr1068), HER-2/neu, AKT and p44/42 (ERK1/2) by cetuximab alone or combined with PD153035. To visualise EGFR in C33A cells, 80 μg of protein were loaded on the SDS–PAGE gels as seen by the higher amount of the endogenous control, HSC-70. (H) Western blotting analysis of the inhibition of phosphorylation of p44/42 (Erk1/2) by cetuximab and PD98059. Student's t-test ^*P<0.05, when compared to control cells. (I) Proposed model of the effects of the combination of cetuximab with chemoradiation or trastuzumab or MAPK pathway inhibitor on CC cell lines. Ligand binding activates signalling through EGFR and triggers the AKT and MAPK pathways. The binding of cetuximab sensitises CC cells to chemoradiation (1) and to trastuzumab (2), leading to cell death independently of EGFR expression levels, but more dependent on EGFR-HER2 signalling. For cells in which activation of the MAPK pathway occurs also through EGFR independent mechanisms, cetuximab inhibition of EGFR sensitises them to PD98059 (3), a MAPK pathway inhibitor, leading to additive effects on the inhibition of cell proliferation.

Figure 4

Effects of cetuximab (100 μg ml⁻¹) alone or combined with trastuzumab (10 μg ml⁻¹), an anti-HER2 MAb, or PD153035 (0.1 μM), an EGFR tyrosine kinase inhibitor, or PD98059 (25 μM), a MAPK pathway inhibitor, on Caski and C33A cells. (A) Effects of cetuximab alone or combined to trastuzumab on the survival in CA. (B) Representative pictures of Caski and C33A cells under cetuximab and trastuzumab treatments in CA. (C and D) Western blotting analysis of the inhibition of EGF-induced phosphorylation of AKT and p44/42 (ERK1/2) by cetuximab alone or combined with trastuzumab in Caski and C33A cells, respectively. (E) Effects of cetuximab alone or combined with PD153035 on cell survival in CA. (F) Effects of cetuximab alone or combined with PD98059 on proliferation by MTT assays. (G) Western blotting analysis of the inhibition of EGF-induced phosphorylation of EGFR (Tyr1068), HER-2/neu, AKT and p44/42 (ERK1/2) by cetuximab alone or combined with PD153035. To visualise EGFR in C33A cells, 80 μg of protein were loaded on the SDS–PAGE gels as seen by the higher amount of the endogenous control, HSC-70. (H) Western blotting analysis of the inhibition of phosphorylation of p44/42 (Erk1/2) by cetuximab and PD98059. Student's t-test ^*P<0.05, when compared to control cells. (I) Proposed model of the effects of the combination of cetuximab with chemoradiation or trastuzumab or MAPK pathway inhibitor on CC cell lines. Ligand binding activates signalling through EGFR and triggers the AKT and MAPK pathways. The binding of cetuximab sensitises CC cells to chemoradiation (1) and to trastuzumab (2), leading to cell death independently of EGFR expression levels, but more dependent on EGFR-HER2 signalling. For cells in which activation of the MAPK pathway occurs also through EGFR independent mechanisms, cetuximab inhibition of EGFR sensitises them to PD98059 (3), a MAPK pathway inhibitor, leading to additive effects on the inhibition of cell proliferation.

Figure 5

EGFR expression of CC cells, induction of ADCC and modulation of VEGF expression by cetuximab (100 μg ml⁻¹) treatment. (A) EGFR mRNA expression by real-time RT-PCR and (B) EGFR cell surface expression measured by flow cytometry. (C) ADCC assay. (D, E and F) VEGF protein concentration detected in the culture medium by ELISA in CC cells. Student's t-test ^*P<0.05, when compared to controls.