Epidermal growth factor receptor signaling is required for microadenoma formation in the mouse azoxymethane model of colonic carcinogenesis

Abstract

Colonic carcinogenesis involves the progressive dysregulation of homeostatic mechanisms that control growth. The epidermal growth factor (EGF) receptor (EGFR) regulates colonocyte growth and differentiation and is overexpressed in many human colon cancers. A requirement for EGFR in colonic premalignancy, however, has not been shown. In the current study, we used a specific EGFR antagonist, gefitinib, to investigate this role of the receptor in azoxymethane colonic premalignancy. The azoxymethane model shares many clinical, histologic, and molecular features of human colon cancer. Mice received azoxymethane i.p. (5 mg/kg/wk) or saline for 6 weeks. Animals were also gavaged with gefitinib (10 mg/kg body weight) or vehicle (DMSO) thrice weekly for 18 weeks, a dose schedule that inhibited normal receptor activation by exogenous EGF. Compared with control colonocytes [bromodeoxyuridine (BrdUrd), 2.2+/-1.2%], azoxymethane significantly increased proliferation (BrdUrd, 12.6+/-2.8%), whereas gefitinib inhibited this hyperproliferation (BrdUrd, 6.2+/-4.0%; <0.005). Azoxymethane significantly induced pro-transforming growth factor-alpha (6.4+/-1.3-fold) and increased phospho-(active) EGFR (5.9+/-1.1-fold), phospho-(active) ErbB2 (2.3+/-0.2-fold), and phospho-(active) extracellular signal-regulated kinase (3.3+/-0.4-fold) in premalignant colonocytes. Gefitinib inhibited activations of these kinases by >75% (P<0.05). Gefitinib also significantly reduced the number of large aberrant crypt foci and decreased the incidence of colonic microadenomas from 75% to 33% (P<0.05). Gefitinib concomitantly decreased cell cycle-regulating cyclin D1 and prostanoid biosynthetic enzyme cyclooxygenase-2 in microadenomas, suggesting that these regulators are key targets of EGFR in colonic carcinogenesis. These results show for the first time that EGFR signaling is required for early stages of colonic carcinogenesis. Our findings suggest, moreover, that inhibitors of EGFR might be useful in chemopreventive strategies in individuals at increased risk for colonic malignancies.

Figure 1

Experimental treatment protocol and growth curve. A, treatment protocol. Mice received azoxymethane (AOM; 5 mg/kg) or saline (azoxymethane vehicle) weekly by i.p. injection for six weeks (arrows). Mice were also treated with gefitinib (10 mg/kg) or DMSO (vehicle) by gavage thrice weekly throughout the study. Animals were sacrificed in week 18. B, growth curves. Weights for the AIN-76A alone and AIN-76A + gefitinib group did not differ from each other and values were combined and presented as control. The azoxymethane-treated and azoxymethane + gefitinib–treated animals showed delayed growth during azoxymethane treatment but resumed normal growth rates afterwards.

Figure 2

Gefitinib inhibits azoxymethane-induced hyperproliferation in mouse premalignant colon. A/J mice were treated as described in Materials and Methods. Two hours before sacrifice, mice received BrdUrd (50 mg i.p./kg body weight). Circular left colon segments were fixed in 10% buffered formalin and BrdUrd incorporation detected by immunostaining as described in Materials and Methods. Representative sections from each group. A, control animals injected with saline (azoxymethane vehicle) and gavaged with DMSO (gefitinib vehicle). B, gefitinib alone. C, azoxymethane alone. D, azoxymethane + gefitinib. Note the increased BrdUrd labeling (black staining) in the azoxymethane alone group compared with animals in the control group. Compared with azoxymethane alone, gefitinib decreased the carcinogen-induced BrdUrd labeling (compare D with C).

Figure 3

Gefitinib inhibits microadenoma formation and pERK and Ki-67 staining. A/J mice were treated as described in Materials and Methods. Mice were sacrificed and colons were harvested after 18 weeks. Colons were fixed flat in formalin and divided longitudinally. Swiss rolls were prepared and embedded in paraffin and sections stained. Representative microadenomas from carcinogen-treated groups. A, pERK in azoxymethane group. Magnification, ×200. B, pERK in azoxymethane + gefitinib group. Magnification, ×200. C, Ki-67 in azoxymethane group. Magnification, ×200. D, Ki-67 in azoxymethane + gefitinib group. Magnification, ×200. Note the decreased proliferation and reduced pERK staining in microadenomas from gefitinib-treated mice. Sections are representative of 11 microadenomas from 7 mice in the azoxymethane alone group and 4 microadenomas from 3 mice in the azoxymethane + gefitinib group.

Figure 4

Gefitinib inhibits azoxymethane-induced EGFR signaling in premalignant mouse colon. A/J mice were treated as described in Materials and Methods. Eighteen weeks after the first azoxymethane injection, mice were sacrificed and colons were flash frozen. Whole-colon lysates were prepared as described in Materials and Methods. A, Western blots of pro-TGF-α, pEGFR, pan-EGFR, pErbB2, pERK, and β-actin expression. A+G, azoxymethane + gefitinib group. B, quantitative densitometry. Fold changes in pro-TGF-α and phospho-active proteins were assessed by densitometry. Four to six animals were analyzed in each group. Values are expressed as fold of control. Columns, mean; bars, SD. ‡ and *, P < 0.05 compared with control; †, P < 0.05 compared with azoxymethane alone.

Figure 5

Gefitinib inhibits cyclin D1 and COX-2 overexpression in microadenomas. A/J mice were treated as described in Materials and Methods. Mice were sacrificed after 18 weeks and colonic Swiss rolls were prepared. Representative sections from carcinogen-treated groups. A, cyclin D1 expression in azoxymethane alone group. B, cyclin D1 expression in azoxymethane + gefitinib group. Cyclin D1 was markedly increased in 7 of 10 tumors from azoxymethane-treated animals, whereas, in the azoxymethane + gefitinib group, staining was comparable with adjacent uninvolved crypts in five of five tumors. C, COX-2 expression in azoxymethane group. D, COX-2 expression in azoxymethane + gefitinib group. COX-2 was increased in 8 of 10 microadenomas from the azoxymethane group compared with one of five microadenomas from the azoxymethane + gefitinib–treated group.