EPHA2-Ephrin-B1 cis-interaction drives an oncogenic reverse signaling, leading to the recurrence of oral cancer

The Eph–Ephrin system classically mediates bidirectional signaling through trans-interactions between adjacent cells, whereas cis-interactions within the same cell are generally thought to attenuate signaling. While receptor-mediated forward signaling suppress tumor growth in certain contexts, the receptor EPHA2 frequently exhibits oncogenic activity through ligand-independent, non-canonical signaling. In contrast, the functional contribution of Ephrin ligands and their reverse signaling in tumor progression remains poorly understood. Paradoxically, high expression of Ephrin ligands—typically considered tumor suppressors—is also linked to poor prognosis via unknown mechanisms. This study uncovers a novel mechanism by which Ephrin-B1 drives oral cancer recurrence through active cis-interaction and reverse signaling. SILAC-based proteomic profiling was performed to identify membrane-associated proteins and pathways enriched in self-renewing oral cancer cells. Biochemical and imaging approaches, including western blotting, flow cytometry, co-immunoprecipitation, FRET-facilitated photoswitching, and proximity ligation assays (PLA), were used to characterize EPHA2–Ephrin-B1 interactions. In vitro kinase assays assessed direct phosphorylation. Functional relevance was evaluated using ALDH1A1-DsRed2 reporter assays, extreme limiting dilution analysis (ELDA), and Ephrin-B1 phosphorylation-deficient mutants. Domain-mapping studies using EPHA2 deletion mutants identified structural determinants of interaction. Clinical relevance was examined using orthotopic xenograft models, TCGA dataset analyses, and tissue microarray-based immunohistochemistry. Self-renewing oral cancer cells exhibited elevated surface expression of EPHA2 and Ephrin-B1, facilitating their cis-interaction within the same cell. This resulted in direct phosphorylation of Ephrin-B1 at Y317 and Y324/329 and subsequent activation of JNK signaling. Disruption of either EPHA2 expression or Ephrin-B1 phosphorylation significantly reduced cancer stem cell (CSC) frequency and tumor initiation capacity. Our data also suggest that EPHA2’s oncogenic role in CSC regulation is mediated, at least in part, through this reverse signaling mechanism. Mechanistically, the FNIII domain of EPHA2 was required for interaction with Ephrin-B1. Clinically, high co-expression of EPHA2 and Ephrin-B1 correlated with poor patient survival and increased recurrence. The highlight of our study is the identification of a novel, active cis-interaction-mediated reverse signaling mechanism in the Eph-Ephrin pathway. Our study discovered Ephrin-B1 as an oncogenic ligand in CSCs. This mechanism, distinct from signaling in normal stem cell niches, offers a potential therapeutic target for oral cancer recurrence.