Prognostic Significance of Endometrioid Carcinoma 1 (ECA1): Enhanced Cancer Cell Proliferation Free

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ECA1 is the human homologue of Notum, a regulator of the Wingless (Wnt/β-catenin) pathway. ECA1 is postulated to act through cleavage of polysaccharide side chains of heparan sulfate proteoglycans, releasing tethered growth factors that may potentiate malignant cell growth via the Wnt pathway. Wnt/β-catenin pathways are frequently activated in colorectal, endometrial, and ovarian carcinomas. We have previously shown that ECA1 transcript levels are increased in endometrioid and ovarian cancers by Northern analysis. To determine whether ECA1 plays a role in cell proliferation, we compared growth rates of vector- and ECA1-transfected HEK 293 cells. Growth rates were determined by measuring cell proliferation at various initial cell densities using the XTT Assay. Our data revealed an approximately two-fold increase in ECA1-transfected HEK 293 cell proliferation compared to vector-control HEK 293 cell. Furthermore, a density-dependence was demonstrated such that growth potentiation was seen only for initial cell densities of 5000 ECA1-transfected HEK 293 cells/well and higher. The density dependence of this effect suggests that a minimum number of ECA1 producing cells were required to produce ECA1 levels associated with growth stimulation. The presence of ECA1 in concentrated condition media and cell lysates were confirmed on Western Blots. We hypothesized that ECA1 induced growth stimulation was linked to the activation of the Wnt/B-catenin pathway. To determine this, ECA1-specific silencing RNA experiments were carried out to decrease ECA1 gene expression and evaluate its effect on cellular proliferation. RT-PCR and Western blot analysis were employed to confirm gene knockdown and decreased protein expression. Cell proliferation was determined using XTT assay and cell viability was determined using trypan blue exclusion. Our data showed a significant decrease in cell proliferation and cell viability for ECA1-transfected cells in the presence of silencing RNA confirming that ECA1 plays a role in increased cell proliferation.