The Proportion of Esophageal Adenocarcinoma Patients With Prior Barrett's Esophagus: Results From a Large Population Based Cohort

Abstract

Introduction:Previous micro array studies and validation with Rt-PCR in a patient cohort from our group have shown that GDF 15 is up regulated in BE. GDF15 is a member of the transforming growth factor-beta superfamily which includes TGF-beta and BMP ligands and may be involved in regulation of tissue differentiation and maintenance. TGF-beta and BMP ligands signal through cell receptors inducing Smad activation. Activated Smad 2/3 builds a complex with Smad 4. The activated Smad-complex accumulates in the nucleus and can initiate gene expression. This transcriptional cascade appears crucial in cell transformation and differentiation as seen in BE. The aim was to examine the signaling potential of GDF15 in this cascade. Methods: Recombinant human GDF15 was used in stimulating Het1a cells (human esophageal squamous epithelial cell line). Smad activation into the nucleus was measured with immunostaining using Smad 1/2/3 antibodies and their nuclear intensity after stimulation. The intensity of activated Smad in the nucleus and quantitative amount of GDF15 utilizing Rt-PCR in Oe 33 cells (esophageal cancer cell line) was measured and then compared with levels in HET1a cells. Results: We found that stimulation of Het1a cells with GDF 15 leads to a time and dose dependant Smad activation. We established that optimal induction of Smad activation in Het1a cells is at 5 ng/ml with GDF15 for 1 h (p<0.0001***). Immunostaining of resting Oe33 cells showed significantly higher levels of activated Smad in the nucleus when compared with resting Het1a cells (p<0.0001***). Further stimulation of Oe33 cells with exogenous GDF15 had no significant effect on Smad activation. Rt-PCR confirmed these findings demonstrating significantly higher GDF15 levels in resting Oe33 cells compared with resting Het1a cells (fold change 111.05; p=0.0031**). Conclusion: We have shown that GDF15, which is up regulated in BE, induces Smad activation and nuclear translocation in a time and dose dependant manner.We also confirmed higher levels of GDF15 in an esophageal cancer cell line with high levels of activated Smad in the nucleus. It is conceivable that GDF15 plays a crucial role in the cell transformation seen in BE through this mechanism. Current work analyses the effects of GDF15 knockdown with siRNA transfection. Future work will examine the affinity of GDF15 to the two known cell receptors of the transforming growth factor superfamily.