Effects of Cancer-Associated EPHA3 Mutations on Lung Cancer

Cancer initiation and malignant progression are multistep processes that involve loss of growth control, evasion of apoptosis, sustained angiogenesis, tissue invasion, and metastasis (1). Developing cancer cells stochastically acquire and selectively accumulate mutations in the genes that encode oncoproteins, tumor suppressors, and their regulators. Receptor tyrosine kinases (RTKs) are important regulators of signal transduction pathways that promote cell growth, survival, invasion, and motility during malignant progression of solid tumors (2). Dysregulation of RTKs, such as EGF receptor family members, by mutation, amplification, or overexpression, can result in increased kinase activity and ultimately oncogenic transformation. The generality of this paradigm of gain-of-function RTK signaling in cancer has been recently challenged by the discovery of the dual roles of the EPH receptors in both promoting and inhibiting oncogenesis and tumor progression in cell lines and mouse models. To date, there has been insufficient evidence from patient outcome-based studies to verify the findings from mechanistic analyses in tumor models. EPH receptors and their membrane-bound ligands, the ephrins, were originally discovered in the 1990s as axonal guidance molecules, and since that time, the EPH proteins have been found to constitute the largest family of RTKs and to be key regulators of cell–cell communication both in development and disease (3,4). The role of EPH receptors in cancer models is complex; they can either promote or inhibit malignancy, depending on ligand stimulation, signaling cross-talk, and other contextual factors (5–8). For example, EPHA2 overexpression is associated with worsened survival in human breast, prostate, and lung cancers and in glioblastoma multiforme (9–17). Overexpression of EPHA2 can induce ligand-independent signaling, resulting in increased tumor cell malignancy in vitro and accelerated tumor growth and metastasis in vivo (18,19). In keeping with these findings, reduced EPHA2 expression in the presence of short interfering RNA or targeted gene deletion inhibited tumor initiation and metastatic progression (19–21). However, ligand-dependent signaling by EPHA2 in both breast cancer and glioblastoma cell lines inhibited their malignant behavior in vitro and tumor growth in vivo (21,22). The conundrum posed by these findings is not completely resolved, and mechanisms that account for these opposing activities are just beginning to be investigated. Recently, “next-generation” DNA sequencing using large cohorts of human lung cancer samples identified various mutations in EPH receptor genes. Notably, somatic mutations in EPHA3, the gene for EPHA3, were present in 5% to 10% of lung adenocarcinomas (23–26). However, those mutations are associated with amino acid substitutions scattered throughout the receptor, and it is unclear whether these nonrecurrent mutations are “driver” or biologically neutral “passenger” genetic alterations. In addition, because EPH receptor signaling can cause either tumor promotion or tumor suppression, the biological impact of somatically mutated variants of EPHA3 in lung cancer remains unclear. In this report, we sought to characterize the functional effects of EPHA3 mutations identified in primary tumors to distinguish between the oncogenic and tumor-suppressive roles of the protein that EPHA3 encodes. We used a combination of genomic and mutational analyses in cell lines and tumor specimens to investigate the role of EPHA3 and its somatic mutations in non–small cell lung cancer (NSCLC). We performed cell proliferation and apoptosis assays in NSCLC cell lines and used mouse xenograft models to assess the function of EPHA3 in vivo. Finally, we identified molecular mechanisms by which EPHA3 regulates tumorigenicity.