P1-12-04: EBP50 – A Novel Biomarker for Resistance to Endocrine and HER2−Targeted Therapies in Breast Cancer.
Authors: Gu G , Covington KR , Fernandez NM , Ando’ S , Fuqua SAW 
Published: June 2012

Background: Therapeutic strategies directed at inhibiting the action of ERa using antiestrogens, such as tamoxifen (Tam), or inhibiting HER2 signalling using a monoclonal antibody-Trastuzumab (Tras) are the standard therapies offered to women with ERa or ERa/HER2−positive cancer. However, not all patients respond to these targeted therapies, and a large number of patients who do respond will eventually develop disease progression or recurrence while on therapy. EBP50 functions as a molecular scaffold to coordinate a diverse range of regulatory processes and a number of studies suggest a role for EBP50 in cancer progression.

Material and Methods: Microarray profiling was used to identify genes associated with response to tamoxifen in primary as compared to metastatic breast tumor recurrences. Stable transfection of EBP50 shRNA was performed in ZR75B and BT-474 cells. Cell response to Tam and/or Tras was evaluated using soft agar and MTT growth assays. The molecular signaling pathways affected by EBP50 knockdown were explored using immunoblot, and immunoprecipitation. Cellular localization was determined using confocal microscopy. Mammosphere potential was used to evaluate the stem cell renewal capacity in BT-474 cells.

Results: The mean levels of EBP50 were reduced in the tamoxifen-resistant, metastatic breast tumors compared to tamoxifen-sensitive tumors. Knockdown of EBP50 levels decreased Tam sensitivity in ZR75B cells. Thus reduction in EBP50 levels analygous to that discovered in resistant patients, conferred resistance to the growth inhibitory effects of Tam. Cells with knockdown of EBP50 (shEBP50 cells) showed higher levels of phosphorylated HER2, EGFR and HER3, as well as their downstream signalling pathways. PI3K and AKT inhibitors were able to restore Tam sensitivity in shEBP50 cells. After long term treatment of ZR75-B cells with Tam resulted in decreased endogenous levels of EBP50, suggesting that only cells evolving with lower EBP50 levels were able to survive Tam treatment. Similarly, knockdown EBP50 in ERa/HER2 positive BT-474 cells significantly increased phosphorylated HER2 and these cells were resistant not only to Tam, but also to Tras treatment. BT-474 cells acquired Tras resistance concomittant with decreased EBP50 levels in a dose-dependent manner respective to parental cells. Interestingly, shEBP50 cells demonstrated an enhanced capacity to form mammospheres compared to vector control cells. We demonstrate that EBP50 is able to bind HER2 using immunoprecipitation, suggesting that EBP50 interacts directly with HER2. Confocal microscope analysis demonstrated the colocalization of these two proteins. Inhibitors to c-Src, PI3K, AKT and EGFR were used in combination with Tras; shEBP50 cells were sensitive to both PI3K and AKT inhibitors, and EGFR inhibitors were able to restore Tras sensitivity.

Discussion: Our data suggest that EBP50 is a novel negative regulator of HER2 signaling, and its loss conferred resistance to both Tam and Tras. EBP50 loss might function to stabilize HER2, and enhance dimerization with EGFR and HER3. We hypothesize that EBP50 levels might be a new predictive biomarker for targeted therapy; patients with low EBP50 levels might best be treated with a combination of therapies including PI3K/AKT inhibitors.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-12-04.