Dual inhibition of vascular endothelial growth factor receptor and epidermal growth factor receptor is an effective chemopreventive strategy in the mouse 4-NQO model of oral carcinogenesis

Abstract

Despite recent therapeutic advances, several factors, including field cancerization, have limited improvements in long-term survival for oral squamous cell carcinoma (OSCC). Therefore, comprehensive treatment plans must include improved chemopreventive strategies. Using the 4-nitroquinoline 1-oxide (4-NQO) mouse model, we tested the hypothesis that ZD6474 (Vandetanib, ZACTIMA) is an effective chemopreventive agent. CBA mice were fed 4-NQO (100 μg/mL) in their drinking water for 8 weeks and then randomized to no treatment or oral ZD6474 (25 mg/kg/d) for 24 weeks. The percentage of animals with OSCC was significantly different between the two groups (71% in control and 12% in the ZD6474 group; P ≤ 0.001). The percentage of mice with dysplasia or OSCC was significantly different (96% in the control and 28% in the ZD6474 group; P ≤ 0.001). Proliferation and microvessel density scores were significantly decreased in the ZD6474 group (P ≤ 0.001 for both). Although proliferation and microvessel density increased with histologic progression in control and treatment cohorts, epidermal growth factor receptor and vascular endothelial growth factor receptor-2 phosphorylation was decreased in the treatment group for each histologic diagnosis, including mice harboring tumors. OSCC from ZD6474-treated mice exhibited features of epithelial to mesenchymal transition, as shown by loss E-cadherin and gain of vimentin protein expression. These data suggest that ZD6474 holds promise as an OSCC chemopreventive agent. They further suggest that acquired resistance to ZD6474 may be mediated by the expression of an epithelial to mesenchymal transition phenotype. Finally, the data suggests that this model is a useful preclinical platform to investigate the mechanisms of acquired resistance in the chemopreventive setting.

Fig. 1

ZD6474-treated animals demonstrate lower proliferative indices and microvessel densities compared to control animals. A, Tissue sections were immunohistochemically stained for Ki-67 and labeling indices (LI) were quantified. The overall LI of the ZD6474 specimens were all significantly lower when compared to the control specimens (p≤0.001). B, Tissue sections were immunohistochemically stained for CD31 and MVD quantified. MVD of the ZD6474 specimens were significantly lower when compared to the controls specimens (p≤0.001). The total group was used for all statistical analysis.

Fig. 2

ZD6474 inhibits the phosphorylation of the EGFR in the 4-NQO model of OSCC. A, tissue sections from control and ZD6474-treated animals were immunohistochemically stained for pEGFR and quantified (B). Expression of pEGFR was significantly lower in the ZD6474-treated specimens when compared to the control specimens (p≤0.001).

Fig. 3

ZD6474 inhibits the phosphorylation of VEGFR-2 in the 4-NQO model of OSCC. A, tissue sections from control and ZD6474-treated animals were immunohistochemically stained for pVEGFR-2 and quantified (B). Expression of pVEGFR-2 was significantly lower in the ZD6474-treated specimens when compared to the control specimens (p≤0.001).

Fig. 4

OSCC arising in ZD6474 treated mice express epithelial to mesenchymal transition (EMT) markers. Tumor samples from control mice demonstrate strong epithelial expression of E-cadherin and stromal expression vimentin. Histologically normal epithelium from the ZD6474-treated animals demonstrate strong expression of E-cadherin and no expression of vimentin (arrows). Conversely, tumor cells from ZD6474 mice demonstrate loss of expression of E-cadherin and strong expression of vimentin.