EGFL6 is a novel HER3 ligand, inducing HER3/integrin heterodimers to induce pERK centrosomal deposition and therapeutic resistance

EGF-like domain multiple-6 (EGFL6) is a secreted tumor growth/migration factor linked with poor outcomes in many tumor types. While EGFL6 is known to signal, in part, via its integrin-binding RGD domain, little else is known about EGFL6 receptors. We evaluated putative EGFL6 receptors and found that EGFL6 treatment of ovarian cancer cells leads to both transient phosphorylation of EGFR and prolonged phosphorylation of HER2 and HER3 and subsequent phosphorylation of ERK (pERK). We found that EGFL6 directly binds HER3. However, EGFL6-driven prolonged activation of HER3 is dependent on an intact EGFL6 integrin-binding RGD domain. Immunoprecipitation and proximity ligation assays confirmed that EGFL6 treatment of cancer cells induces HER2/3-integrin-β3 heterocomplexes. Suggesting EGFL6 could play a role in resistance to HER targeting therapies, EGFL6 is upregulated in EGFR/HER receptor inhibitor-resistant cells, and EGFL6 treatment increases resistance to EGFR/HER inhibitors in vitro. Interestingly, we found that, in EGFL6-treated ovarian cancer cells undergoing mitosis, pERK localizes to the centrosome. Both EGFL6-neutralizing antibodies and HER protein-targeted inhibitors resulted in aberrant pERK centrosomal localization with associated altered mitotic spindle alignment and mitotic catastrophe. Furthermore, combination anti-EGFL6 therapy with the pan-EGFR receptor inhibitor neratinib, compared to either therapy alone, led to an increase in aberrant pERK localization and cancer cell death in vitro and significant restricted tumor growth in vivo. Combined, our data suggests that EGFL6 is a new ligand for HER3 and that dual targeting of the EGFL6/HER signaling axis, via altered pERK localization, may be an effective therapeutic strategy in ovarian cancer. SIGNIFICANCE: This work reveals that EGFL6 is a previously unrecognized ligand for HER3 which can increase resistance to HER family-targeted therapy. We also reveal a novel function of pERK downstream of pHER3 at the centrosome in mitosis. Importantly, we show that EGFL6 is an important therapeutic target to enhance the efficacy of EGFR/HER-targeted therapy.