Cancer invasiveness plays a major role in the mortality of patients with solid tumors, and deregulated cell adhesion and migration are suspected to drive invasive behavior. Since Eph receptor tyrosine kinases control both cell attachment and migration, they may act to define the level of cancer invasiveness. EphB6 is an unusual Eph receptor, lacking catalytic capacity due to alterations in its kinase domain. Interestingly, increased metastatic activity is associated with reduced EphB6 receptor expression in several tumor types, including breast cancer. This emphasizes the potential of EphB6 to act as a suppressor of cancer aggressiveness; however, the mechanism of its action is not well understood. We show that restoration of EphB6 expression in invasive breast cancer cells supports actin-dependent spreading and attachment and blocks invasiveness. EphB6 stimulation induces its tyrosine phosphorylation, which is crucial for its function and is mediated by the EphB4 receptor. This is accompanied by EphB6-c-Cbl interaction and phosphorylation of c-Cbl partner, the Abl kinase. Cbl silencing suppresses Abl phosphorylation, cell adhesion, and morphologic changes and blocks the ability of EphB6 to inhibit invasiveness, confirming its importance for EphB6 activity. Despite its crucial role in EphB6 responses, EphB4 also acts in an EphB6-independent manner to enhance invasive activity, suggesting that cancer invasiveness may be defined by the balance in the EphB6-EphB4 system. Overall, our observations suggest a new role for EphB6 in suppressing cancer invasiveness through c-Cbl-dependent signaling, morphologic changes, and cell attachment and indicate that EphB6 may represent a useful prognostic marker and a promising target for therapeutic approaches.

The nonhomologous end-joining (NHEJ) pathway is essential for the preservation of genome integrity, as it efficiently repairs DNA double-strand breaks (DSBs). Previous biochemical and genetic investigations have indicated that, despite the importance of this pathway, the entire complement of genes regulating NHEJ remains unknown. To address this, we employed a plasmidbased NHEJ DNA repair screen in budding yeast (Saccharomyces cerevisiae) using 369 putative nonessential DNA repair-related components as queries. Among the newly identified genes associated with NHEJ deficiency upon disruption are two spindle assembly checkpoint kinases, Bub1 and Bub2. Both observation of resulting phenotypes and chromatin immunoprecipitation demonstrated that Bub1 and -2, either alone or in combination with cell cycle regulators, are recruited near the DSB, where phosphorylated Rad53 or H2A accumulates. Large-scale proteomic analysis of Bub kinases phosphorylated in response to DNA damage identified previously unknown kinase substrates on Tel1 S/T-Q sites. Moreover, Bub1 NHEJ function appears to be conserved in mammalian cells. 53BP1, which influences DSB repair by NHEJ, colocalizes with human BUB1 and is recruited to the break sites. Thus, while Bub is not a core component of NHEJ machinery, our data support its dual role in mitotic exit and promotion of NHEJ repair in yeast and mammals.T he repair of DNA double-strand breaks (DSBs) is an essential process required for the preservation of genome integrity and the normal functioning of the cell (1). These cytotoxic lesions are repaired by major DSB repair pathways, including the homologous recombination (HR) (2) and nonhomologous end-joining (NHEJ) systems (1). While the former is the prevalent pathway in the unicellular budding yeast Saccharomyces cerevisiae (3), the latter is more prevalent in mammalian cells, especially those that are quiescent (4), and can repair DNA lesions even if there is no homologous strand (5). Notably, the impairment of NHEJ in mammalian cells is frequently linked to genomic instability, cancer, and lymphoid V(D)J (i.e., variable, diversity, and joining gene segments) recombination defects. Therefore, a detailed molecular understanding of this pathway would provide critical insight into the genetic risk factors related to carcinogenesis or immunological disorders (6).As in mammalian cells, the core components of the classical NHEJ pathway in S. cerevisiae depends on three major complexes, YKu (Ku), MRX, and DNL4, which are rapidly recruited to DSBs (7). Initially, the yeast Ku heterodimer (Ku70/80) binds to each end of a DSB, serving as an anchor for protein complexes involved in securing and annealing the break, also suppressing the competing HR pathway (8). After this, the DSB processing complex MRX (Mre11-Rad50-Xrs2), which acts at an early stage of both the NHEJ and HR repair pathways (9), spans the lesions so that the DNA ligase complex, DNL4 (i.e., Dnl4-Lif1-Nej1), can rejoin the DSB ends (10).While the actions of these core protein complexes in y...