Dual inhibition of both the epidermal growth factor receptor and erbB2 effectively inhibits the promotion of skin tumors during two-stage carcinogenesis

Abstract

The erbB family of receptor tyrosine kinases are known to play important roles in normal epithelial development and epithelial neoplasia. Considerable evidence also suggests that signaling through the epidermal growth factor receptor (EGFR) plays an important role in multistage skin carcinogenesis in mice; however, less is known about the role of erbB2. In this study, to further examine the role of both erbB2 and EGFR in epithelial carcinogenesis, we examined the effect of a dual erbB2/EGFR tyrosine kinase inhibitor, GW2974, given in the diet on skin tumor promotion during two-stage carcinogenesis in wild-type and BK5.erbB2 mice. In BK5.erbB2 mice, erbB2 is overexpressed in the basal layer of epidermis and leads to heightened sensitivity to skin tumor development. GW2974 effectively inhibited skin tumor promotion by 12-O-tetradecanoylphorbol-13-acetate in wild-type and BK5.erbB2 mice, although a more marked effect was seen in BK5.erbB2 mice. In addition, this inhibitory effect was reversible when GW2974 treatment was withdrawn. GW2974 inhibited 12-O-tetradecanoylphorbol-13-acetate-induced epidermal hyperproliferation, which correlated with reduced activation of both the EGFR and erbB2. These results support the hypothesis that both the EGFR and erbB2 play an important role in the development of skin tumors during two-stage skin carcinogenesis, especially during the tumor promotion stage. Furthermore, the marked sensitivity of BK5.erbB2 mice to the inhibitory effects of GW2974 during tumor promotion suggest greater efficacy for this compound when erbB2 is overexpressed or amplified as an early event in the carcinogenic process.

Figure 1

Treatment protocols for dietary administration of GW2974 in Experiments #1 and #2 of the current study. In Experiment #1 each diet group had between 22–25 mice, and in Experiment #2 each diet group had between 22–24 mice.

Figure 2

Effects of GW2974 on skin tumor promotion in wild-type and BK5.erbB2 mice. (A) Number of tumors per mouse and (B) tumor incidence in Experiment #1. In Experiment #1, two groups of homozygous transgenic and two groups of wild-type mice were initiated with DMBA (50 nmol), followed two weeks later with twice-weekly applications of TPA (6.8 nmol).□, BK5.erbB2 mice fed AIN76A control diet; ■, BK5.erbB2 mice fed AIN76A diet containing 200 ppm GW2974; ○, Wild-type mice fed AIN76A control diet; ●, Wild-type mice fed AIN76A diet containing 200 ppm GW2974. (C). Average number of tumors per mouse after diet switch in wild-type mice (Wt) and BK5.erbB2 mice (Tg) in Experiment #1. □, BK5.erbB2 mice that were switched from GW2974 to AIN76A control diet; ■, BK5.erbB2 mice that were fed continuously with diet containing GW2974; ◐, Wild-type mice that were switched from GW2974 to AIN76A control diet; ●, Wild-type mice that were fed continuously with diet containing GW2974.

Figure 3

Effect of GW2974 on skin tumor promotion in wild-type and BK5.erbB2 mice in Experiment #2. (A) Number of tumors per mouse in Experiment #2. □, BK5.erbB2 mice fed AIN76A control diet; ■, BK5.erbB2 mice fed AIN76A diet containing GW2974; ○, Wild-type mice fed AIN76A control diet; ●, Wild-type mice fed AIN76A diet containing GW2974. (B) Number of tumors per mouse after diet switch in wild-type (Wt) and BK5.erbB2 mice in Experiment #2. ◐, Wild-type mice that were switched from GW2974 to AIN76A◧ control diet; ●, Wild-type mice that were fed continuously with diet containing GW2974; , BK5.erbB2 mice that were switched from GW2974 to AIN76A control diet; ■, BK5.erbB2 mice that were fed continuously with diet containing GW2974.

Figure 4

Tumor size (diameter) and categorization by size at the end of Experiment #2. Tumor diameters were measured in week 18 at the end of Experiment #2. ■, tumors > 2 mm in diameter; □, tumors ≤ 2 mm in diameter. * p < 0.05

Figure 5

H&E and BrdU stained sections of skin from wild-type and BK5.erbB2 mice treated with various diets in Experiment #2. Skins were collected at the end of Experiment #2 (i.e., at week 18).

Figure 6

Western blot analysis of the levels of total and phosphorylated erbB2 and EGFR in the dorsal skin. (A) Skin from wild-type mice fed with AIN76A or GW2974. (B) Skin from BK5.erbB2 mice fed with AIN76A or GW2974 diet. A group of four mice received four topical applications (given twice-weekly) of either 6.8 nmol TPA or acetone over 2 weeks and were sacrificed 4 h after the last treatment. Mice were fed with either AIN76A control diet or GW2974 containing diet from 2 weeks prior to the first TPA or acetone treatment until the end of the experiment. (a) Representative Western blots. (b) Relative expression levels measured via densitometry normalized to β-actin levels. Note that the Western blot of p-erbB2, erbB2 and p-EGFR from wild-type mice needed significantly longer exposure time to develop the film compared to erbB2 mice. *p <0.05

Figure 7

Immunostaining for erbB2 and p-erbB2 in the skin of wild-type mice and BK5.erbB2 mice fed with AIN76A and GW2974 diets. Skin was collected from the experiment shown in Figure 6. Upper panels: Skin from a wild-type mouse on AIN76A diet treated with acetone. Middle panels: Skin from a BK5.erbB2 mouse on AIN76A diet treated with acetone. Lower panels: Skin from BK5.erbB2 mice on GW2974 diet treated with acetone.