Undergraduate Dominican University of California SANTA ROSA, California, United States
Genna Roan (Dominican University of California)| Grace Alexander (Dominican University of California)| Klarisse Cruz (Dominican University of California)| Janelle Nguyen (Dominican University of California)| Mary Sevigny (Dominican University of California)
Cyclooxygenases (COXs) are enzymes that catalyze the formation of prostaglandins. Prostaglandins are a class of lipid mediators that can promote inflammation, pain, and fever as well as maintain a variety of normal physiological functions. Cyclooxygenases exist in two forms, COX-1 and COX-2, both of which are localized to the luminal membrane of the endoplasmic reticulum. COX-2 expression is usually induced in response to certain stimuli, such as infection. However, overexpression of COX-2 has been correlated to numerous cancers, such as breast, colon and prostate cancer. COX-2 exists as two major glycoforms of 72 and 74kDa, the latter resulting from an additional oligosaccharide chain at amino acid residue Asn580. Past studies from our lab have shown that this glycosylation regulates the COX-2 protein turnover in the cell. The proteins E-cadherin— a tumor suppressor— and b-catenin—a tumor driver— are often regulated by COX-2 expression. The purpose of this study is to determine if the glycosylation of COX-2 at Asn580 affects the downstream expression of E- cadherin and b-catenin and subsequent migratory potential in MCF-7 breast cancer cells. MCF-7 cells were transiently transfected with either the wild-type or Asn580-mutant COX-2 gene or empty plasmid (control). RNA isolated from cells was subjected to semi-quantitative RT-PCR, and cell lysates were analyzed via western blotting following by immunostaining. Migratory potential was determined three days after transfection using in vitro transwell cell migration assays. No differences were found in migratory potential amongst the three cell groups. We found that E-cadherin expression at both the transcriptional and translational levels was reduced in cells expressing the mutant COX-2 gene for 72 hours. Interestingly, transcriptional expression of b-catenin was also reduced after 48 and 72 hours of expression of the mutant COX-2 gene, though protein expression appeared unchanged. These results indicate that although no changes in migratory potential could be discerned three days after transfection, glycosylation of COX-2 at Asn580 did decrease the expression of both the tumor suppressor E-cadherin and the tumor driver gene β-catenin in the MCF-7 breast cancer cell line, thus obscuring the role of COX-2 in breast cancer progression.