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THE ESTROGEN-RELATED RECEPTOR ALPHA (ERRalpha) PROMOTES CELL PROLIFERATION IN DLD-1 COLORECTAL CANCER CELL LINE
T Lassalle, G Bernatchez, V Giroux, JC Carrier
Canadian Institutes of Health Research Team on Digestive Epithelium, Département d’Anatomie et Biologie Cellulaire, Faculté de Médecine et des Sciences de la Santé, Université de Sherbrooke, Sherbrooke, Québec
The orphan nuclear receptor ERRalpha, which share sequence similarity with estrogen receptors, in mainly recognized for its role in oxidative metabolism. In addition, ERRalpha gene expression has been shown to be increased in colon cancer compared to matched normal mucosa. Moreover, it has been found that a splicing variant of ERRalpha, in which the fifth exon is missing, is predominantly expressed in normal compared to cancer tissues. Skipping of the fifth exon removes ERRalpha’s transcriptional coactivator binding site but leaves its dimerization region intact. The aims of this study were to evaluate the proliferative potential of the full-length form and characterize the delta5ERRalpha splice variant in colonic epithelial cells.
METHODS: The expression of full-length ERRalpha and delta5ERRalpha has been determined in normal and cancerous intestinal cell lines. Seconds, we have generated two stable DLD-1 colon cancer cell lines using lentiviral-mediated gene transfer: one in which ERRalpha have been silenced using short hairpin (sh) RNA targeting ERRalpha and the other in which the delta5ERRalpha splice variant has been re-expressed.
RESULTS: First and foremost, we observed high ERRalpha expression, but no delta5ERRalpha, in various colon cancer cell lines. In contrast, low levels of both ERRalpha and delta5ERRalpha were detected in normal intestinal cells. We confirmed by co-immunoprecipitation that delta5ERRalpha dimerizes with the full-ength form but is unable to interact with the main coactivator of ERRalpha, PPAR-gamma Coactivator 1 alpha (PGC-1alpha). As expected, delta5ERRalpha does not have transcriptional activity on ERRE-luciferase genes and acts as an ERRalpha dominant negative variant. The efficient silencing of ERRalpha expression in DLD-1 cells significantly reduced their growth rate. In addition, tumor growth in immunocompromised mice bearing the inoculated sh-ERRalpha-DLD-1 was significantly less compared with mice bearing the empty vector DLD-1. Similarly, delta5ERRalpha expression in DLD-1 cells resulted in significant reduction of cell and tumor growth.
CONCLUSION: Using a shRNA as well as a naturally occurring dominant negative strategy, we provided evidence that ERRalpha promotes cell growth in DLD-1 colorectal cancer cell line.