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

Although EPHA2 is a receptor tyrosine kinase widely expressed in many cancers, it exhibits a uniquely high frequency of coding sequence mutations in cholangiocarcinoma, a cancer of the biliary tract with dismal prognosis. EPHA2 is extensively studied, but very little is known about the role of EPHA2 cancer mutations. To define the functional significance of EPHA2 mutations in biliary tract cancers, we generated representative EPHA2 mutants and monitored major receptor autophosphorylation sites as indicators of kinase activity-dependent signal transduction (known as forward signaling). We found that missense mutations in the ligand-binding domain abrogate ephrinA ligand binding, while missense mutations in the kinase domain abrogate kinase activity. The effects of missense mutations in other domains were less pronounced and varied depending on the phosphosite. The majority of the EPHA2 mutations are nonsense or frame-shift mutations that introduce early stop codons. They generate EPHA2 truncated forms that lack an intact kinase domain or, in some cases, most of the coding sequence. Several EPHA2 mutants tested inhibited tyrosine phosphorylation of co-expressed EPHA2 wild-type, indicating the ability to exert dominant negative effects. We show that EPHA2 forward signaling in cholangiocytes inhibits the ERK oncogenic pathway and cell proliferation, suggesting that loss-of-function mutations facilitate tumor development in the biliary tract. Indeed, an EPHA2 kinase-inactive mutant, but not EPHA2 wild-type, induced proliferative masses consistent with well differentiated cholangiocarcinoma in a validated mouse model of cholangiocarcinogenesis. Thus, EPHA2 has the attributes of a driver gene with tumor suppressor activity in biliary tract cancers.