Dual targeting of EphA2 and ER restores tamoxifen sensitivity in ER/EphA2-positive breast cancer.

Abstract Abstract #3020 Background: Overexpression and altered function of EphA2 receptor tyrosine kinase are critical in the progression of breast cancer and provide a novel target for breast cancer therapy. We have previously demonstrated that EphA2 overexpression decreases estrogen dependence and tamoxifen sensitivity both in vitro and in vivo. EA5, a novel monoclonal antibody that mimicks the binding of ephrin A to EphA2, reverses the effect of EphA2 overexpression and restores tamoxifen sensitivity in EphA2-transfected MCF-7 cells (MCF-7EphA2) in vitro. Here we report the impact of EA5 on in vivo tumor growth and its ability to overcome in vivo tamoxifen resistance in MCF-7EphA2 xenografts. Furthermore, we investigated the mechanisms by which EphA2 overexpression decreases the estrogen dependence and contributes to tamoxifen resistance in ER+ breast cancer models.
 Material and Methods: MCF-7 cells transfected with vector (MCF-7neo) or EphA2 (MCF-7EphA2) were implanted in the right and left mammary fat pads of athymic mice. Treatment with EA5 (5 mg/kg/i.p./5 days/week), vehicle, tamoxifen (1 mg/oral gavage/5 days/week), or EA5 and tamoxifen in combination was initiated once tumors were established. To explore the role of EphA2 overexpression on ER-dependent mechanisms, we used two different ER+/EphA2-transfected cell line models (MCF-7neo/ MCF-7EphA2 and T47Dneo/ T47DEphA2). We measured ER activity and expression of ER-dependent proteins in response to 17β-estradiol (E2;10-10 M), 4-hydroxy-tamoxifen (10-6 M, and EA5 antibody (3μg/mL). Interaction of ER and EphA2 was investigated using GST-pulldown and co-immunoprecipitation approaches. Gene array studies suggested that EphA2 may impact ER via noncanonical pathways, leading to an investigation of focal adhesion kinase (FAK) signaling.
 Results: EA5 inhibits primary tumor growth and restores tamoxifen sensitivity in the MCF-7EphA2 in vivo model;EA5 had no impact on in vivo tumor growth in MCF-7neo xenografts. Using T47DEphA2 in vitro model, we verified that EphA2 decreases ER activation in response to E2 stimulation consistent with our earlier results in MCF-7EphA2 model. We found no direct interaction between ER and EphA2 and no difference in expression of canonical ER-dependent proteins, ER coactivators or corepressors. However, E2 stimulation phosphorylates FAKTyr925 in ER+/EphA2+ cell lines but not in ER+/EphA2- cell lines. Treatment of T47DEphA2 cells with EA5 and tamoxifen leads to dephosphorylation of FAKTyr925 in the presence of E2 stimulation.
 Conclusion: Our data demonstrate that dual targeting of EphA2 and ER is a promising approach for delaying resistance to tamoxifen. The data support our hypothesis that EphA2 impacts ER function via a FAK dependent pathway. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 3020.