In vitro activity: CA3 potently inhibits esophageal adenocarcinoma cell growth in vitro. CA3 can effectively suppress tumor cell proliferation, induce apoptosis, reduce tumor sphere formation, and the population of ALDH1+ cells. CA3 specially inhibits Tead/YAP1 transcriptional activity but shows no inhibitory activity on other transcriptional factors-Super-TOP/Wnt, CBF1/Notch, and AP-1 after cotransfection of their respective individual promoter luciferases in 293T cells. CA3 preferentially inhibits CSC properties enriched in radiation-resistant esophageal adenocarcinoma cells.

Kinase Assay: The SOX9 luciferase reporter was described previously. The 5 × -UAS-luciferase reporter and Gal4-TEAD4 constructs, also described previously, were obtained from Dr. Johnson of MD Anderson Cancer Center. Transient cotransfection esophageal adenocarcinoma cells with the SOX9 luciferase reporter and a Renilla vector or 5 × -UAS-luciferase reporter and Gal4-TEAD4 with a CMV-β-gal construct was performed as described previously.

Cell Assay: The esophageal adenocarcinoma cells and their resistant counterparts were treated with 0.1% DMSO (control), CA3 at different doses. For combination treatment experiments, treatment of the cells with CA3, 5-FU, or a combination at different concentrations was administered for 6 days as indicated, and the cell viability was assessed using an MTS assay as described previously. All assays were performed in triplicate and repeated at least three times.