br Materials and Methods br Results br Discussion

Materials and Methods
Results
Discussion Although widely accepted as a prognostic biomarker in advanced CRC, our findings in this study clearly showed that EGFR protein expression was dramatically elevated in colonic adenomatous polyps in FAP patients, even at a very early stage. More importantly, a mechanistic study indicated that the widespread overexpression of EGFR might occur as a consequence of COX-2 activation, a common pathological event in various gastrointestinal precancerous lesions. Coupled with the fact that the absence of Egfr reduced intestinal polyps by 90% in mice (Roberts et al., 2002), these results indicated that COX-2 might drive colon tumorigenesis, at least in partly, through up-regulation of EGFR, which phenotypically facilitates neoplastic cell transformation in precancerous lesions. As such, EGFR might be a novel target for CRC chemoprevention. Previously, PGE2, the major COX-2-derivated PG, was reported to be capable of transactivating the EGFR kinase cascade in colon cancer flt3 inhibitor (Pai et al., 2002), whereas activation of EGFR could conversely stimulate COX-2 biosynthesis (Coffey et al., 1997). Taken together, a positive feedback loop could possibly exist between COX-2 and EGFR in intestinal tumorigenesis. In this regard, FAP patients whose colonic polyps express high levels of both COX-2 and EGFR might be the most likely to benefit from a combination anti-COX-2/EGFR therapy (Torrance et al., 2000). COX-2 is known to exert its biological function through its derivate prostaglandins. If this is the case, identifying the specific prostaglandins that act downstream of COX-2 would be essential for understanding how COX-2 activation enhances EGFR expression. The most direct experimental approach is by examining PGs\' biosynthesis upon COX-2 inhibition. However, the biosynthesis of all five of the major bioactive PGs\' in primary intestinal adenoma epithelial cells was suppressed by aspirin or celecoxib treatment (Supplementary Fig. 1). An exception is the effect of aspirin on PGI2 production. The interpretation of such phenomenon is still incomplete. Thus, further studies examining susceptibility to intestinal polyps in mice with targeted deletions in specific prostaglandin synthases and receptors are needed. Knowing the specific cell type that expresses COX-2 would be essential for understanding how COX-2 promotes CRC progression. However, no consensus exists at present as to which cell types within a colon tumor specifically express COX-2. Some groups, including us, found that COX-2 is primarily expressed in the epithelial cells of colon adenomas and sporadic human colon cancers (Sano et al., 1995). Another group has reported COX-2 expression in the stromal compartment of polyps from mice (Oshima et al., 1996). We are uncertain whether these differences in COX-2 localization are due to experimental artifacts or simply because of inherent variability within the sample. Within a given tumor, COX-2 may also be expressed in more than one cell type (Masferrer et al., 2000). The interpretation of this phenomenon is still pending. Although our findings are interesting, several questions remain unanswered. For example, one caveat is that the doses of aspirin and celecoxib used in in vitro studies are higher than their clinically relevant concentration. Although aspirin exposure decreases the elevated EGFR levels in FAP patients, in the majority of individuals who were aspirin users or non-users, the levels of EGFR were still overlapping. This is consistent with the outcome of CRC chemoprevention in which aspirin benefits most but not all of the overall population. Another issue is that our sample size is small, and data collection as well as the final conclusion might be limited only to colonic adenoma in FAP patients. Although COX-2 and EGFR were co-localized in colon adenocarcinomas from FAP patients, a functional association between them was not observed (Supplementary Fig. 2A, B). Moreover, knockdown of COX-2 didn\'t or only weakly affected the levels of EGFR in 4 colon cancer cell lines (Supplementary Fig. 2C). All of these findings suggested that colon cancer cells have already escaped from COX-2 dependence (Lev-Ari et al., 2007), but the mechanism of action remains unclear.