Drug repurposing with a better understanding of the underlying mechanism has provided new avenues to find treatment for malignancies. Esophageal adenocarcinoma (EAC) is a rapidly increasing cancer with a dismal 5-year survival rate of <15%. Lack of efficient treatment options contributes to the high mortality rate of EAC. To find new therapy against EAC we performed unbiased drug screening of an FDA-approved drug library and identified that the cardiac glycosides including Ouabain, Digoxin and Digitoxin efficiently inhibit the proliferation of EAC cell lines (OE33 and OE19) both in vitro and in vivo. RNA-Sequencing analysis combined with RNAi screening revealed that Ouabain suppresses the proliferation of EAC cells through downregulation of p38 MAP-Kinase 6 (MAP2K6, also known as MKK6). Consistently, shRNA-mediated knockdown of MKK6 reduced the proliferation of EAC cells and tumor growth. Further analysis demonstrated that MKK6 inhibition leads to the reduced levels of the transcription factor SOX9. In line with this finding, deletion of SOX9 with CRISPR/Cas9 resulted in decreased proliferation of EACs in 3D organoid culture and reduced tumor growth. Together these findings establish a druggable axis that can be harnessed for therapeutic gain against EAC.

重新利用药物以更好地了解其潜在机制为寻找治疗恶性肿瘤的方法提供了新途径。食道腺癌（EAC）是一种快速增长的癌症，其5年生存率低于15％。缺乏有效的治疗选择会导致EAC的高死亡率。为了找到针对EAC的新疗法，我们对FDA批准的药物库进行了公正的药物筛选，并确定了包括Ouabain，Digoxin和Digitoxin在内的强心苷在体外和体内均能有效抑制EAC细胞系（OE33和OE19）的增殖。。RNA测序分析与RNAi筛选相结合表明，哇巴因通过下调p38 MAP激酶6（MAP2K6，也称为MKK6）抑制EAC细胞的增殖。一致地，shRNA介导的MKK6的敲低减少了EAC细胞的增殖和肿瘤的生长。进一步的分析表明，MKK6抑制导致转录因子SOX9的水平降低。与该发现一致，用CRISPR / Cas9删除SOX9导致3D类器官培养物中EAC的增殖减少并降低了肿瘤的生长。这些发现共同建立了一个可药物化的轴，可以利用该轴来获得针对EAC的治疗收益。