Frequent EPHA2 receptor mutations in cholangiocarcinoma disrupt receptor forward signaling supporting a tumor suppressor role

ABSTRACT EPHA2 is a receptor tyrosine kinase highly expressed in many cancers. By analyzing cancer patient databases for mutations in the EPHA2 coding sequence, we found that cholangiocarcinoma (a hepatobiliary cancer with dismal prognosis) exhibits a uniquely high incidence of EPHA2 mutations. To deUine the functional signiUicance of these mutations, we generated representative EPHA2 mutants and monitored major receptor autophosphorylation sites as indicative of kinase activity-dependent signal transduction (known as forward signaling). We found that missense mutations in the ligand-binding domain abrogate ligand binding and ligand-induced EPHA2 tyrosine phosphorylation, while most missense mutations in the kinase domain abrogate kinase activity. We detected less pronounced effects of missense mutations in other domains, which vary depending on the phosphosite, suggesting that these EPHA2 mutations might differentially affect (or bias) different downstream signaling pathways. Other EPHA2 mutations introduce early stop codons and encode receptor truncated forms lacking all or part of the kinase domain. We also found that an EPHA2 secreted truncated form, a transmembrane truncated form, and a full-length kinase inactive mutant can inhibit tyrosine phosphorylation of co-expressed EPHA2 wild-type. Taken together, these data suggest that mutations interfering with EPHA2 forward signaling facilitate cholangiocarcinoma development. We indeed obtained evidence that an EPHA2 kinase inactive mutant, but not EPHA2 wild-type, can induce proliferative masses consistent with well differentiated cholangiocarcinoma in a validated mouse model of cholangiocarcinogenesis. Taken together, our Uindings suggest that EPHA2 is a driver gene in cholangiocarcinoma and that its forward signaling has tumor suppressor activity.