Saturday, May 2, 2015

Aspirin Prevents Colorectal Cancer by Inhibiting EGFR Kinase

Aspirin intake reduces the risk of colorectal cancer (CRC), but the molecular mechanisms remain unknown. Based on data from various articles, researches and literature, Cyclooxygenase-2 (COX-2) has long been suspected to be the primary target for aspirin and NSAID-mediated CRC chemoprevention. There are many studies that either support or deny COX-2 as the primary target, but it is well established that COX-2 activation alone is insufficient to cause tumorigenesis, evidenced by the fact that COX-2 transgenic mice fail to develop tumors spontaneously. Collectively, COX-2 might function as a tumor promoter rather than as an initiator, but the mechanism of action by which COX-2 drives tumorigenesis remains imperfectly understood.

The epidermal growth factor receptor (EGFR), a transmembrane receptor tyrosine kinase of the ErbB family, was elevated in up to 80% of CRC cases and is associated with clinical outcomes.

Researchers demonstrated that COX-2 might drive intestinal tumorigenesis by up-regulating EGFR expression in familial adenomatous polyposis (FAP) patients, an effect that could be attenuated by regular aspirin use. This is the first instance where a kinase activity is being linked with aspirin, and a possible molecular mechanism for CRC inhibition is postulated using both a kinase as well as COX-2 enzyme.

Study and Data

Familial adenomatous polyposis (FAP) patients were recruited by the Gastroenterology and Hepatology group at Mayo Clinic, Rochester, Minnesota. Through a protocol approved by the Mayo Clinic Rochester MN IRB, all subjects with FAP seen at Mayo Clinic Rochester MN between January 1990 through May and from whom polyp tissue was available were identified through a search of clinical diagnoses and pathology reports in the electronic medical record (EMR). The EMR of the identified FAP cases with available tissue (n = 178) was searched to identify those with a history of NSAID use at the time that the available polyp tissue was collected. Cigarette smoking and inflammatory bowel diseases are independent risk factors for CRC, while FAP patients suffering hypertension or cardiovascular diseases might take additional anticoagulant drugs other than aspirin. Thus, exclusion criteria also included cigarette smoking, inflammatory bowel diseases, hypertension, a history of cardiovascular disease, and pregnancy and subjects were not on any other pharmacological treatments. FAP patients who reported taking two or more standard (325 mg) aspirin tablets per week within the previous 12 months were classified as regular aspirin users (n = 25) and those reporting consumption of less aspirin were classified as aspirin nonusers (n = 25).  Individuals in the healthy control group (n = 25) were normal subjects who underwent colonoscopy screening. The gender ratio in each group was approximately 1:1.

The experimental assays included determination of prostaglandin production, electrophoretic mobility-shift assay, rt-pcr analysis, reporter assays, western blot analysis abd fianlly statistical analysis.


The following results are reported:

1. COX-2 and EGFR are Positively Correlated in FAP Patients:  Data strongly suggested that EGFR and COX-2 were co-localized in the polyp tissues in FAP patients. Relationship between COX-2 and EGFR expression in colonic adenomatous polyps from FAP patients  indicated that in 95% of the FAP cases (19/20) exhibiting COX-2 overexpression, EGFR was also highly expressed. Further quantification analysis indicated that the observed expression levels of COX-2 and EGFR were not independent of each other but were highly and positively correlated (p = 0.0015; R = 0.7585) in adenomatous polyps from FAP patients.

2. Functional Relevance of COX-2 in the Regulation of EGFR: Data from immunohistochemistry analysis reveal that COX-2 and EGFR were highly expressed in intestinal adenoma epithelial cells, but not in normal intestinal epithelial cells. Furthermore, treatment with either aspirin or celecoxib, a well-known COX-2 inhibitor, lowered EGFR expression in intestinal adenoma epithelial cells.  Murine Embryonic Fibroblasts (MEFs) with COX-2 gene deficiency (COX-2-/-) have a much lower EGFR level. Furthermore, typical COX-2 activators, including inflammatory cytokine interleukin-1 beta (IL-1b) and bacterial lipopolysaccharide (LPS), dramatically boosted both COX-2 and EGFR protein expression in COX-2 wildtype cells compared to knockout cells.

3. COX-2 Modulates EGFR Transcription Through Activator Protein-1 (AP-1): Researchers hypothesized that COX-2 might modulate EGFR at the transcriptional level by activating the Egfr promoter.  Aspirin was previously reported as an inhibitor of the activity of activator protein-1 (AP-1), one of the potential transcription factors for Egfr. Results from the study indicated that AP-1 activity as well as AP-1 DNA binding activity was markedly attenuated in the absence of cox-2 gene expression.

Issues Still Not Solved

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. Although COX-2 and EGFR were co-localized in colon adenocarcinomas from FAP patients, a functional association between them was not observed.

The authors conclude as “In summary, this study revealed a previously unknown functional association between COX-2 and EGFR during colorectal carcinogenesis, and provided an explanation as to how aspirin intake can lower the risk of CRC in FAP patients.

Article citation: Li, H.; et. al. Aspirin Prevents Colorectal Cancer by Normalizing EGFR Expression, EBioMedicine 2015. 10.1016/j.ebiom.2015.03.019