Cyclooxygenase-2 and prostaglandin E 2 (PGE 2 ) levels are increased in colorectal cancers and a subset of adenomas. PGE 2 signaling through the EP4 receptor has previously been associated with colorectal tumorigenesis. However, changes in EP4 expression during adenoma to carcinoma progression have not been investigated, neither has whether levels of EP4 influence important markers of malignant potential, such as anchorage-independent growth or the tumors growth response to PGE 2 . We report using immunohistochemistry that in vivo EP4 receptor protein expression was increased in colorectal cancers (100%) as well as adenomas (36%) when compared with normal colonic epithelium. EP4 expression was also higher in colorectal carcinoma compared with adenoma cell lines and increased with in vitro models of tumor progression. Adenoma (PC/AA/C1 and RG/C2) and carcinoma cell lines (HT29) were growth stimulated by PGE 2 up to 0.5 Mmol/L. However, although carcinoma and transformed adenoma (PC/AA/C1SB10C, a transformed derivative of PC/AA/C1) cells remain stimulated by higher doses of PGE 2 (10 Mmol/L), the adenoma cell lines were inhibited. Interestingly, enforced expression of EP4 in the adenoma cell line, RG/ C2, resulted in stimulation of growth by 10 Mmol/L PGE 2 and promoted anchorage-independent growth. Both in vivo and in vitro data from this study suggest that increased EP4 receptor expression is important during colorectal carcinogenesis. We propose that high levels of PGE 2 in a tumor microenvironment would select for cells with increased EP4 expression, and that the EP4 receptor may therefore represent an important target for colorectal cancer prevention and treatment. (Cancer Res 2006; 66(6): 3106-13)

Overexpression of the growth factor receptors EGFR and erbB2 occurs frequently in several human cancers and is associated with aggressive tumour behaviour and poor patient prognosis. We have investigated the effects of ZD1839 (Iressa), a novel EGFR tyrosine kinase inhibitor, on the growth, in vitro and in vivo, of human cancer cell lines expressing various levels of EGFR and erbB2. Proliferation of EGFRoverexpressing A431 and MDA-MB-231 cells in vitro was potently inhibited (50%-70%) by ZD1839 with half-maximally effective doses in the low nanomolar range. EGFR (also known as erbB1) is the prototypic member of the erbB family of receptor tyrosine kinases, whose other members are erbB2, erbB3 and erbB4. 1 Amplification of the EGFR or erbB2 gene, leading to protein overexpression, occurs frequently in a number of human cancers, particularly those derived from breast, lung, ovary and prostate. 2 In addition, many tumours produce growth factors that drive autocrine or paracrine proliferation via these pathways. 3 Overexpression of EGFR or erbB2 is usually associated with more aggressive tumour behaviour, resistance to chemotherapies and poor clinical outcome. The underlying reasons for this have been partially elucidated by numerous studies on the mechanisms of erbB receptor activation and signal transduction. 1 Binding of EGF or EGF-like ligands to the EGFR promotes receptor dimerisation, increased tyrosine kinase activity and autophosphorylation of the cytoplasmic domain of the receptor on specific tyrosine residues. These residues then serve as docking sites for molecules including Grb2, PLC and Shc, which trigger multiple signals whose targets control biologic responses such as proliferation. For example, both Grb2 and Shc facilitate activation of the Ras-ERK MAP kinase pathway, whereas PLC-␥1 generates the second messengers diacylglycerol and inositol trisphosphate, which lead to protein kinase C activation and elevation of intracellular calcium, respectively. Numerous studies in various biologic systems indicate that these and other signalling pathways are critical for full mitogenic responsiveness to EGF. 4 With the exception of erbB2, which has no identified ligand, erbB receptor family members are activated by distinct ligands, each possessing a unique subset of binding sites for the recruitment of signalling molecules. It is now well established that erbB receptors are capable of forming heterodimers. Heterodimerisation alters ligand specificity and affinity and increases the complexity of the downstream signals generated. erbB2 appears to act as the preferred heterodimeric partner for each of the other 3 erbB subtypes. 5 Indeed, evidence suggests that erbB2-containing heterodimers have greater affinity for the relevant ligand, 6 generate more prolonged signals and enhance the biologic effects of erbB ligands such as EGF. 5,7 In addition, overexpression of erbB2 is thought to favour the formation of erbB2 homodimers that signal in the absence of ligand. 8 Because of their frequent overexpression in human c...