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.

Triple-negative breast cancer (TNBC) tumours that lack expression of oestrogen, and progesterone receptors, and do not overexpress the HER2 receptor represent the most aggressive breast cancer subtype, which is characterised by the resistance to therapy in frequently relapsing tumours and a high rate of patient mortality. This is likely due to the resistance of slowly proliferating tumour-initiating cells (TICs), and understanding molecular mechanisms that control TICs behaviour is crucial for the development of effective therapeutic approaches. Here, we present our novel findings, indicating that an intrinsically catalytically inactive member of the Eph group of receptor tyrosine kinases, EPHB6, partially suppresses the epithelial–mesenchymal transition in TNBC cells, while also promoting expansion of TICs. Our work reveals that EPHB6 interacts with the GRB2 adapter protein and that its effect on enhancing cell proliferation is mediated by the activation of the RAS-ERK pathway, which allows it to elevate the expression of the TIC-related transcription factor, OCT4. Consistent with this, suppression of either ERK or OCT4 activities blocks EPHB6-induced pro-proliferative responses. In line with its ability to trigger propagation of TICs, EPHB6 accelerates tumour growth, potentiates tumour initiation and increases TIC populations in xenograft models of TNBC. Remarkably, EPHB6 also suppresses tumour drug resistance to DNA-damaging therapy, probably by forcing TICs into a more proliferative, drug-sensitive state. In agreement, patients with higher EPHB6 expression in their tumours have a better chance for recurrence-free survival. These observations describe an entirely new mechanism that governs TNBC and suggest that it may be beneficial to enhance EPHB6 action concurrent with applying a conventional DNA-damaging treatment, as it would decrease drug resistance and improve tumour elimination.