Model of Colonic Carcinogenesis Microadenoma Formation in the Mouse Azoxymethane Epidermal Growth Factor Receptor Signaling Is Required for Updated

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 F 1.2%], azoxymethane significantly increased proliferation (BrdUrd, 12.6 F 2.8%), whereas gefitinib inhibited this hyperproliferation (BrdUrd, 6.2F 4.0%; <0.005). Azoxymethane significantly induced pro-transforming growth factor-A (6.4 F 1.3–fold) and increased phospho(active) EGFR (5.9 F 1.1–fold), phospho-(active) ErbB2 (2.3 F 0.2–fold), and phospho-(active) extracellular signal-regulated kinase (3.3 F 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. [Cancer Res 2007;67(2):827–35] Introduction Colonic carcinogenesis involves a stepwise accumulation of mutations in tumor suppressor genes and proto-oncogenes (1). These mutations in turn dysregulate mechanisms controlling crypt cell proliferation, maturation, and apoptosis that are normally controlled by multiple homeostatic mechanisms, including signals from the epidermal growth factor (EGF) receptor (EGFR; ref. 2). EGFR belongs to the ErbB family of receptor tyrosine kinases (RTK) that also includes ErbB2, ErbB3, and ErbB4. Normal colonocytes express EGFR, ErbB2, and ErbB3 (3–5). ErbB receptors have an extracellular domain that binds ErbB ligands with high affinity, a single transmembrane domain, and an intracellular domain. The intracellular domain contains a tyrosine kinase and multiple effector domains. With ligand binding, the ErbB receptors form homodimeric or heterodimeric complexes. This dimerization activates the intrinsic RTK that in turn phosphorylates in trans multiple tyrosine residues in the cytoplasmic receptor tail. These autophosphorylation sites, and adjacent residues, create domains that recruit adapter and effector proteins that propagate downstream signals (2). Downstream pathways include Ras/ extracellular signal-regulated kinase (ERK) and phosphatidylinositol 3-kinase/AKT (2). Targets of these signaling pathways include cyclin D1 and cyclooxygenase-2 (COX-2; refs. 6–8). Cyclin D1 is an important EGFR target that controls G1-S cell cycle progression (9). COX-2 is the key inducible and rate-limiting enzyme required for prostaglandin biosynthesis (10). Multiple ligands for ErbB receptors have been described. Some ligands bind to unique ErbB receptors, whereas others can bind to multiple ErbB receptors. The diverse combinations of ErbB ligands and receptors afford multiple levels of control by these potent growth-regulating signals (2, 11). In human colon cancers, alterations in ErbB receptors, including up-regulation of EGFR and ErbB2, and increases in EGFR ligands, including transforming growth factor-a (TGF-a), have been described (4, 12, 13). Increases in EGFR or ErbB2 expression portend greater invasiveness of these tumors and a worse prognosis (14, 15). Cyclin D1 and COX-2 are also up-regulated in human and experimental colon cancers (8, 16, 17). Although studies indicate that several ErbB receptors are overexpressed in many advanced human colon cancers, inhibitors or antibodies to these receptors alone possess limited clinical efficacy (18). Presumably, many tumors have acquired activating mutations downstream of EGFR or exploited other growthpromoting signals, rendering EGFR signaling redundant. In contrast to the relatively small number of established colon Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). Requests for reprints: Marc Bissonnette, Department of Medicine, University of Chicago Hospitals and Clinics, MC 4076, 5841 South Maryland Avenue, Chicago, IL 60637. Phone: 773-702-9898; Fax: 773-702-2182; E-mail: mbissonn@medicine. bsd.uchicago.edu. I2007 American Association for Cancer Research. doi:10.1158/0008-5472.CAN-05-3343 www.aacrjournals.org 827 Cancer Res 2007; 67: (2). January 15, 2007 Research Article American Association for Cancer Research Copyright © 2007 on February 23, 2013 cancerres.aacrjournals.org Downloaded from DOI:10.1158/0008-5472.CAN-05-3343